Abstract
Background/Aim: Low selectivity and high frequency of side-effects are the major problems of currently used chemotherapeutics. Among natural compounds, the polyprenylated acylphloroglucinol, guttiferone E, isolated from Brazilian red propolis, has attracted attention due to its marked anticancer properties and was evaluated here for its role against osimertinib-resistant H1975 cells (with double mutations of epidermal growth factor receptor: EGFR L858R/T790M). Materials and Methods: Guttiferone E was obtained from red propolis using established extraction procedures. Guttiferone E was tested using the H1975 cell line in in vitro (2D and 3D) cell cultures and in vivo in BALB/c athymic nude mice. Live/dead assay was also performed to support the results. Tumor tissues obtained from in vivo studies were used for western blotting. Guttiferone E reduced H1975 cell viability in a concentration-dependent manner. The IC50 values in 2D and 3D cell lines were 2.56±0.12 μM and 11.25±0.34 μM. Furthermore, at 10 mg/kg intraperitoneally, guttiferone E significantly reduced the tumor volume in tumor xenografts when used alone and in combination with carboplatin. Guttiferone E and carboplatin displayed synergistic inhibition of H1975 cells and animal tumors. Co-treatment of guttiferone E with carboplatin induced more prominent apoptosis than treatment with either drug alone. Guttiferone E treatment induced cleavage of poly-ADP ribose polymerase and induced apoptosis by significantly reducing levels of mammalian target of rapamycin, sirtuin 1, sirtuin 7, superoxide dismutase, programmed death-ligand 1, and programmed cell death 1 in tumor tissues. Conclusion: Our results show guttiferone E to be a promising, novel and potent antitumor drug candidate for osimertinib-resistant lung cancer with EGFR L858R/T790M mutations.
Lung cancer is among the most lethal malignancies in the world, with an elevated rate of mortality and morbidity caused by an abnormal growth of cells that infiltrate tissues and organs (1). It is the second prime cause of cancer death in both men and women in the United States. According to the American Cancer Society, in 2023, it was estimated that over 238,340 people (117,550 men and 120,790 women) would be diagnosed with lung cancer, out of whom 127,070 would die from this disease (2). Lung cancer presents a wide range of symptoms and signs since it can develop at numerous places in the bronchial tree. More than 70% of individuals with lung cancer have stage III or IV advanced disease when first diagnosed (3). Surgery, radiation therapy, and chemotherapy are the most often used approaches to manage this malignancy. Chemotherapeutic drugs target both cancerous and healthy cells, leading to major unpleasant side effects such as diarrhea, hair loss, mucositis, immunosuppression, nausea and vomiting, which lower the quality of life for patients (4). For this reason, chemotherapeutic drugs need to be formulated using various nanotechnologies including antibody conjugates, liposomes, and exosomes (5-9).
A significant proportion of the population in developing nations uses natural products to treat medical conditions because they are typically associated with a lower chance of causing adverse side-effects (10). Natural products are also one of the best sources for discovering new bioactive molecules and as a starting point for obtaining new derivatives by synthetic or semi-synthetic strategies (11). In the past decade, we, along with many researchers across the globe, have tried to evaluate natural compounds against various cancer types and have shown their good anticancer potential (7, 12-15). Propolis, a natural resinous product, is produced by bees, predominantly Apis mellifera L. It has a variety of chemical components that vary depending on its geographic and botanical origins, such as climatic conditions, plant resources, origin, and the period that the bees collected the raw materials of propolis (16). The primary botanical source of Brazilian red propolis (BRP) and other red propolis types described worldwide is Dalbergia ecastaphyllum L. Taub. (Fabaceae). BRP comprises isoflavonoids, flavonoids, and pterocarpans (17). Recently, a second botanical source, Symphonia globulifera L. (Clusiaceae), was identified and was observed to be responsible for giving BRP a distinct phytochemical profile compared to other red propolis types. S. globulifera provides polyprenylated benzophenones, adding outstanding biological activities to BRP (18).
We reported a class of exciting and understudied secondary metabolites, known as polycyclic polyprenylated acylphloroglucinols, represented by guttiferone E in a mixture with its position isomer xanthochymol (19, 20). The majority of guttiferones have been isolated from plants belonging to the Clusiaceae family’s Garcinia genus, though reports of guttiferones from other genera within the same family have also been made, including S. globulifera L. Besides the botanical sources, chemical structures and biological activities are diverse from BRP (21, 22). Guttiferone E/xanthochymol exhibit biological activities previously reported in the literature, including anti-inflammatory (23, 24) anti-HIV (25), cytotoxic (26, 27) antineoplastic (28), and antitumor properties (29). Targeting the toll-like receptors/interleukin-1 receptor-associated kinase 1 pathway and inhibiting downstream nuclear factor kappa-light-chain-enhancer of activated B cells (NF-
B) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling are two means contributing to the anti-inflammatory properties of guttiferone E (19, 30). Human colon cancer cell lines (HCT 116 and HT 29), oral carcinoma cell line (KB), and a variety of drug-sensitive/resistant phenotypic cancer cells (breast MDA-MB-231-BCRP cells) have all been examined for the possible anticancer properties of guttiferone E (26, 27, 31). The cytotoxic mechanism of action of guttiferone E in these tumor cells includes involvement with mitochondrial membrane potential, rise in expression of genes such as DNA damage-induced transcript 3, as well as X-box binding protein 1 and activating transcription factor 4, with subsequent triggering of the endoplasmic reticulum apoptotic pathway in addition to the expression of apoptosis-inducing enzymes caspase 8, caspase 9, and caspase 3/7 (26, 27).
The prevailing gold standard of cancer treatment combines radiotherapy with chemotherapy, such as platinum-based regimens. Cisplatin, the most widely used chemotherapeutic drug, has activity against various malignancies (32). An alternative therapeutic option for cancer of the ovary, testis, head, neck, and non-small-cell lung cancer (NSCLC) is carboplatin (33). Carboplatin is less toxic than cisplatin but must often be administered in larger doses. It has been utilized in combination therapy with other drugs or as a mono-therapeutic in treating a variety of malignancies (34, 35). Like other platinum-based drugs, carboplatin acts by interacting with deoxyribonucleic acid, the cell’s genetic material (36). However, cancer resistance to platinum-based drugs represents a significant setback (37). Finding an alternate chemotherapeutic or complementary lung cancer treatment plan is necessary to combat carboplatin resistance and lessen its adverse effects. Using natural compounds or their derivatives in conjunction with carboplatin is one of the most promising methods.
Lung cancer carcinogenesis and development are intimately linked to epidermal growth factor receptor (EGFR) mutation and overexpression, making EGFR an essential target for lung cancer treatment (38, 39). In order to attach to the ATP-binding site of EGFR tyrosine kinase and stop its autophosphorylation, small-molecule inhibitors have been created. However, their inability to have therapeutic effects is mainly a result of drug resistance and toxicity (40, 41). It is challenging to use fourth-generation EGFR-tyrosine kinase inhibitors when more diverse EGFR mutations (T790M and L858R) result in drug resistance (42, 43), which pose a significant barrier to treating lung cancer. Therefore, it is critical to develop a novel treatment strategy to solve the drug-resistance issue. Hence, in this study, we evaluated the cytotoxic potential of guttiferone E in osimertinib-resistant lung cancer, using the H1975 cell line and tumor xenografts, in combination with carboplatin. Further, the possible mechanism of action of the combination was also investigated.
Materials and Methods
Materials. BRP was acquired from the COAPER Beekeepers’ Association’s apiaries in Canavieras, Bahia, Brazil. Carboplatin, obtained from Sigma-Aldrich (St. Louis, MO, USA), met good manufacturing practice and good laboratory practice standards. Osimertinib was purchased from Medchem Express (Monmouth Junction, NJ, USA). Additional materials and reagents were supplied by Sigma-Aldrich. Primary and secondary antibodies were provided by Cell Signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Paso Robles, CA, USA). RPMI-1640 media and H1975 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). H1975 cells were grown in RPMI-1640 media with antibiotics (PSN; GIBCO, Grand Island, NY, USA) and 10% fetal bovine serum (FBS; Bio-Techne, Minneapolis, MN, USA).
To induce resistance, H1975 cells were subjected to a range of osimertinib doses (0.5 nM to 10 nM) for a period of 6 months. The resulting H1975R cells were utilized in fewer than 15 passage cultures and kept in a regulated, humidified incubator with 5% CO2 at 37°C.
Collection and processing of BRP for extract preparation. BRP was acquired in June 2021. It was crushed and macerated for extraction using a 7:3 (w/v) ethanol-to-water ratio. The BRP was subjected to a 24-h resulting process in a shaker incubator with 120 rpm agitation, with five repetitions. The resultant extracts were lyophilized to create a dry product, subsequently concentrated under the influence vacuum in a Büchi rotary evaporator. The material was then refrigerated between 2 and 8°C in a properly sealed container.
Yield of production and isolation of benzophenones from BRP. The initial BRP mass before extraction (IM) and the material recovered after extraction and solvent removal (EM) were used to assess the yield of the extracted material as a percentage of mass obtained according to the following equation:
The BRP extract served as the source for benzophenones (derivatives of benzoyl phloroglucinol). Using a solid-phase technique, the extract was partitioned by combining 300 g of microcrystalline cellulose with 100 g of extract. Using solvents of increasing polarity, the blend was poured onto a glass column (13 11 cm2 i.d.): ethyl acetate (2 l), hexane (2 l), dichloromethane (2 l), and ethanol (98%, 2 l). Then, the fractions were lyophilized after being vacuum-concentrated. Vacuum liquid chromatography separated the hexane fraction (30 g) using silica gel 60 (300 g, 40-60 mesh ASTM) as the stationary phase and a mixture of hexanes and ethyl acetate as the mobile phase in a glass column. This process resulted in four sub-fractions. Fraction FH2, with a higher yield of extracted material, was submitted for a purification step through semi-preparative HPLC.
Chromatographic analysis, purification, and identification. A Shimadzu (Kyoto, Japan) semi-preparative high-performance liquid chromatography (HPLC) instrument with an SPD-20 A UV/vis detector was used to purify benzoyl phloroglucinol derivatives from BRP. The extract, fractions, and isolated substances were subjected to HPLC studies for purification utilizing an HPLC Waters 2695 chromatographer with a 2998 PDA (Waters Corporation, Milford, MA, USA) and Ascentis Express C18 column (2.7 m, 150 4.60 mm2; Supelco; Sigma-Aldrich). A Synergi Polar-RP column (4 m, 250 21.5 mm; Phenomenex; Sigma-Aldrich) was selected, with methanol (solvent A) and acid-water (0.1:99.9, solvent B) and a flow rate of 10 ml/min, respectively, as the mobile gradient phases.
For the analysis, gradient conditions for phase B were 20-50% for 40 min, 50-100% for 90 min, 100% for 95 min, and 20% for 100 min. The flow rate was 1 ml/min. To gather mass spectrometric data, direct injection was performed on an orbitrap mass spectrometer (Thermo Scientific) in negative ionization mode. The gradient conditions of phase B for purification of substances from hexane fractions were as follows: 85% for 25 min, 100% 30 min, 100-85% for 35 min, and 85% for 37 min.
Nuclear magnetic resonance (NMR) spectra of the isolated compounds were collected on a Bruker-Avance spectrometer (Bruker, Billerica, MA, USA) running at 500 MHz for 1H and 125 MHz for 13C NMR, respectively. The substances were dissolved in 1% trifluoroacetic acid (TFA)-deuterated methanol (CD3OD). The NMR results were compared with information from literature in order to verify the structure of the isolated substances.
In vitro cytotoxicity analysis. Guttiferone E and carboplatin were dissolved in dimethyl sulfoxide. MTT assay was used to conduct 2D cytotoxicity assays. Briefly, osimertinib-resistant H1975R cells were seeded in 96-well plates (5×103 cells/well) and were treated with different concentrations (1-100 μM) of guttiferone E and carboplatin alone for 48 h to determine the half-maximal inhibitory concentrations (IC50). In another set of experiments, we performed a cytotoxicity assay using the IC25 of carboplatin in combination with guttiferone E (1-100 μM) as a therapy for 48 h, followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described elsewhere (44).
Magnetic nanoshuttle 3D cytotoxicity assay. The cell pellet of osimertinib-resistant H1975 cells was obtained after passage. A solution with NanoShuttle-PL magnetic beads (Greiner Bio-One, Monroe, NC, USA) consisting of gold, iron and poly-L-lysine (10 μl for 100,000 cells) was added in a complete medium (RPMI 1640 with 10% FBS). After mixing for 1-2 min, the suspension was differentially centrifuged three times for 5-6 min at 200 × g. After centrifugation, the cell suspension with the nanoshuttle solution was supplemented with the necessary growth medium (enough for 96 wells). Then, approximately 15,000 cells were plated into a 96-well plate using a spheroid drive. The plate was incubated for 3 days at 37°C with 5% CO2. The medium was changed on day 3. On day 5, the cells were finally given a 48 h treatment as in the 2D cytotoxicity assay. To assess cytotoxicity, the medium was aspirated, and then the MTT test was performed. After that, the percentage of untreated control cells was used to calculate the relative cell viability (45).
Fluorescent staining by dual acridine orange/ethidium bromide (AO/EB). Osimertinib-resistant H1975 cells were plated in 3D culture using magnetic nanoshuttle solution at a density of 15,000 cells in 96-well plates and incubated at 37°C for 24 h in a CO2 incubator. The 3D spheroid cells were treated with guttiferone E (7.5 μM, 12.5 μM, and 17.5 μM), carboplatin (300 μM, 400 μM, and 500 μM) and their combination (with the IC25 concentration of carboplatin, 200 μM) and further incubated for 48 h. The concentrations chosen were based on the IC50 range of the drugs. Subsequently, the medium was removed from the wells, and the cells were washed with saline. After that, the cell spheroids were dipped in 300 μl of staining solution (100 μg/ml AO and 100 μg/ml EB) mixed with PBS, mixed thoroughly, and then incubated for 10 min. Finally, the cells were washed again, and a Nikon Eclipse Ti 100 inverted fluorescence microscope (Nikon Instruments, Inc., Melville, NY, USA) was used to obtain images. The images were assessed for quantification using Image J 1.36 analytical software (National Institutes of Health, Bethesda, MD, USA).
In vivo tumor studies. BALB/c athymic nude mice (male and female, aged 6 weeks) were obtained from Envigo (Indianapolis, IN, USA) and housed in American Association for Accreditation of Laboratory Animal Care-accredited facilities at Florida A&M University, where stringent pathogen-free conditions were maintained. The mice were accommodated in standard enclosures kept at 37°C and 60% relative humidity. All experimental procedures adhered strictly to the guidelines of the Florida A&M University Institutional Animal Care and Use Committee (approval number: 023-02), ensuring ethical treatment and a 1-week acclimation period before the commencement of experiments. Matrigel (1:1) was combined with osimertinib-resistant H1975 cells (4×106) which were then subcutaneously implanted into each mouse’s right flank. Ten days after implantation, mice were randomly assigned to four groups, each consisting of five animals: Untreated, guttiferone E, carboplatin, and guttiferone E plus carboplatin combination. Intraperitoneal injections of guttiferone (10 mg/kg), carboplatin (25 mg/kg), or their combination were administered every other day, following dosages established from previous literature (45, 46). Tumor growth was monitored over a 10-day treatment period using digital Vernier calipers, and tumor volumes were calculated using the formula TV=½ × length × width2, where ‘length’ and ‘width’ represent the largest and perpendicular dimensions of the tumors, respectively (47).
Western blotting: The tumor tissue samples were homogenized in T-PER buffer supplemented with phosphatase and protease inhibitors (1:100 dilution; Sigma-Aldrich) on ice for 30 min, followed by centrifugation at 10,000 × g for 20 min to collect the clear supernatant for protein estimation. Protein concentrations were determined, and 20-40 μg of protein was loaded onto sodium dodecyl sulfate polyacrylamide gels for electrophoresis. The separated proteins were then transferred onto nitrocellulose membranes and blocked with 3% bovine serum albumin for 90 min to prevent non-specific binding. Overnight incubation with primary antibodies specific to poly-ADP ribose polymerase (PARP) (cat no. 9542S), sirtuin 7 (SIRT7) (cat no. 5360S), sirtuin 1 (SIRT1) (cat no. 9475S), programmed death-ligand 1 (PD-L1) (cat no. 13684S), programmed death 1 (PD-1) (cat no. 86163S), mechanistic target of rapamycin (mTOR) (cat no. 2972S) (all at 1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA), SOD2 (cat no. SC30080; Santa Cruz Biotechnology, Paso Robles, CA, USA), and β-actin (cat no. 4970L; 1:1000 dilution; Cell Signaling Technology) was performed. After primary antibody incubation, membranes were washed with Tris-buffered saline with Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit cat no. 7074S, anti-mouse cat no. 7076S; 1:3,000 dilution; Cell Signaling Technology) for 2 h at room temperature. Protein bands were visualized using enhanced chemiluminescence, and densitometric analysis of relative band densities was conducted using Image J 1.36 software [National Institutes of Health (48-51)].
Statistical analysis. All data are presented as the mean±standard deviation. The significance of the differences between the treatment groups was assessed using either Student’s t-test or one-way analysis of variance followed by Bonferroni’s Multiple Comparison Test in GraphPad Prism version 10.0 (GraphPad, Boston, MA, USA). Statistically significance was accepted when p-values were less than 0.05.
Results
Isolation, purification, and characterization. The hydroalcoholic extraction of BRP furnished 520 g of lyophilized extract, accounting for approximately 52% of the starting material. The solid-phase partitioning of the alcoholic extract using solvents of increasing polarity yielded 24.9 g (hexanes), 54.8 g (dichloromethane), 5.4 g (ethyl acetate), and 4.9 g (ethanol) of each fraction, respectively. The isolation focused on the polycyclic polyprenylated acylphloroglucinols was predominantly observed in the hexane fraction. The fractionation and subsequent purification processes followed previously described methods for BRP chemical analysis (18). After submitting 800 mg of a polycyclic polyprenylated acylphloroglucinol-enriched hexane fraction (FH2) to final purification by preparative HPLC, a final yield of 300 mg of guttiferone E/xanthochymol was obtained; HRESIMS negative mode m/z 601.3546 [M – H] – (calcd. for C38H50O6, 601.3529), 525.3232, 183.0116, 109.0284.
The analytical method using HPLC-DAD, described in the previous section, was employed to determine the purity of the isolates. A 100-min elution was required to separate guttiferone E/xanthochymol and oblongifolin B, which depended on this extended organic phase range to elute other compounds in BRP extract (Figure 1): Guttiferone E/xanthochymol: yellow amorphous powder; HRESIMS negative mode m/z 601.3546 [M – H] – (calcd for C38H50O6, 601.3529); UV λmax=245, 354 nm. NMR 1H (500 MHz, CD3OD and 1% of TFA): 7.20 (d, J=2.1 Hz, 1H), 6.99 (dd, J=8.2; 2.1 Hz, 1H), 6.72 (d, J=8.8 Hz, 1H), 5.09-5.00 (m, 2H), 4.89-4.84 (m, 1H), 4.50 (s, 1H), 4.46 (d, J=9.6 Hz, 1H), 2.71 (dd, J=13.0; 9.5 Hz, 1H), 2.57 (m, 2H), 2.57 (m, 2H), 2.26 (ax, d, J=14.0 Hz, 1H), 2.14-2.07 (m, 1H), 2.05-1.99 (m, 4H), 1.95-1.79 (m, 2H), 1.73 (s, 3H), 1.69 (s, 3H), 1.68 (s, 3H), 1.60 (s, 3H), 1.57 (d, J = 1.0 Hz, 2H), 1.64 (s, 6H), 1.49 (s, 4H), 1.16 (s, 3H), 0.98 (s, 6H). NMR 13C (125 MHz, CD3OD and 1% of TFA): 210.6 (C-9), 196.2 (C-1), 195.6 (C-10), 194.3 (C-3), 152.3 (C-14), 149.3 (C-31), 146.1 (C-13), 135.8 (C-19), 133.5 (C-26), 132.6 (C-36), 129.5 (C-11), 125.4 (C-25), 125.1 (C-16), 124.0 (C-35), 121.2 (C-18), 119.3 (C-2), 117.3 (C-12), 115.1 (C-15), 112.5 (C-32), 69.7 (C-4), 59.8 (C-8), 50.2 (C-5), 47.9 (C-6), 45.1 (C-30), 43.8 (C-7), 36.7 (C-29), 33.4 (C-34), 30.2 (C-24), 27.4 (C-23), 27.0 (C-17), 26.4 (C-20), 26.0 (C-37), 25.5 (C-27), 23.2 (C-22), 18.3 (C-21), 18.2 (C-28), 18.1 (C-33), 17.8 (C-38).
High-performance liquid chromatography profile and chemical structure of guttiferone E. Chromatographic profile of (A) Brazilian red propolis extract and (B) its isolate guttiferone E/xanthochymol. (C) Chemical structure of guttiferone/xanthochymol.
Effect of guttiferone E on 2D and 3D cell viability. The study investigated the cytotoxic effects of guttiferone E and carboplatin using viability assays conducted on osimertinib-resistant H1975 cells exposed to different concentrations of these compounds over a 48-h period (Figure 2). Guttiferone E exhibited notable cytotoxicity, with IC50 values of 2.56±0.12 μM in 2D cell cultures and 11.25±0.78 μM in 3D cell culture. In contrast, carboplatin had considerably higher IC50 values of 195.2±9.77 μM in 2D and y393.58±11.26 μM in 3D cell culture (Table I).
Effect of guttiferone E and carboplatin, alone and in combination, on the viability of osimertinib-resistant H1975 cells in 3D culture. Viability relative to untreated control cells is shown. The results are the mean±standard deviation (n=3).
Half-maximal inhibitory concentration (IC50) values of guttiferone E, carboplatin, and their combination, against osimertinib-resistant H1975 cells in 2D and 3D culture.
Dose reduction indices for both guttiferone E and carboplatin are given in Table II. Additionally, the combined effect of guttiferone E with carboplatin was assessed, revealing a synergistic interaction of carboplatin with guttiferone E, as indicated by combination index values <1, indicating, synergism in both 2D and 3D cell culture (Table II). The IC50 values for guttiferone E in combination with carboplatin were 1.21±0.10 μM in 2D and 3.02±0.21 μM in 3D cell culture (Table I). These findings underscore the potential of combining guttiferone E with carboplatin to enhance their anticancer effects, particularly in multidimensional cell models.
Dose reduction index (DRI) and combination index (CI) values for guttiferone E and carboplatin at non-constant ratio combinations.
Effect of the combination of guttiferone E and carboplatin on apoptosis. To assess cell viability and distribution of live and dead cells in 3D cultures after 48 h of incubation, a live/dead assay using dual AO/EB staining was conducted on H1975R cells and examined under a fluorescent microscope. Live cells and early apoptotic cells were identified by their uptake of AO, emitting green fluorescence, whereas late apoptotic and necrotic cells took up EB, emitting red fluorescence. The images revealed a significant decrease in the number of live cells with increasing concentrations of carboplatin and guttiferone E. In the control group, cells remained predominantly alive at 24 hours but showed a hypoxic core with dead cells by 48 h of incubation. Overall, the images illustrated that guttiferone E exhibited greater cytotoxicity compared to carboplatin, and the combination of guttiferone E and carboplatin resulted in the lowest relative mean cell viability (p<0.001). These findings highlight the potent cytotoxic effects of guttiferone E and its potential synergistic interaction with carboplatin in reducing cell viability in 3D cell cultures (Figure 3).
Fluorescence images of dual acridine orange/ethidium bromide staining assay. The experiment investigated the impact of guttiferone E (Gut-E) and carboplatin (Carbo), alone and in combination (Combo; with 200 μM of carboplatin) on the viability of osimertinib-resistant H1975 cells in 3D culture. Live cells fluoresce green, while dead cells fluoresce red in the merged images. The scale bar on each image represented 200 μm. B: The graph depicted the relative mean cell viability based on the live/dead dual staining assay results, presented as the mean±standard deviation (n=3). Statistical significance is indicated as: *p<0.05, **p<0.01, ***p<0.001 vs. the control, and ^^p<0.01, ^^^p<0.001 vs. single treatments.
Effect of guttiferone E and carboplatin on an osimertinib-resistant H1975 xenograft model in athymic nude mice. The effect of guttiferone E, carboplatin, and their combination on tumor volume at various treatment days was examined in female/male athymic nude mice and was contrasted with the control group. After the first day of treatment, we noticed no discernible difference between the tumor volumes in the various groups. By the fourth day of treatment, there was a statistically significant difference in tumor volumes among the groups, particularly evident when compared to the control group. Treatment with 25 mg/kg carboplatin, 10 mg/kg guttiferone E, and their combination led to a marked reduction in tumor size (p<0.01). The tumor volume in athymic nude mice was also considerably reduced after 10 days of treatment by guttiferone E (p<0.01), carboplatin (p<0.01), and their combination (p<0.001). Compared to the control group, it was observed that treatment with guttiferone E alone resulted in a substantial decrease in tumor volume, but the effect was increased when it was paired with carboplatin (Figure 3). Our results demonstrate that even at a dose of 10 mg/kg, guttiferone E reduced the tumor burden in osimertinib-resistant H1975 xenografts as compared to treatment with double the dose of carboplatin, and the combination of both therapies significantly reduced tumor volume (p<0.001) (Figure 4). This outcome underscores the efficacy of carboplatin and guttiferone E individually, as well as their synergistic effect when administered together, in inhibiting tumor growth within this timeframe of observation.
Guttiferone E and carboplatin, individually and combined, were observed to reduce tumor volumes in athymic nude mice bearing osimertinib-resistant H1975 tumors. The bar graph illustrates tumor volume over a 10-day period following treatment with carboplatin, guttiferone E, and their combination. The data are mean values with error bars representing the standard deviation (n=5). Statistical significance is indicated as: *p<0.05, **p<0.01, ***p<0.001, and ^p<0.05 vs. single treatments.
Effect of guttiferone E and carboplatin combination on osimertinib-resistant H1975 tumor xenografts on the PARP–PD-1–PD-L1 axis. We evaluated the anti-apoptotic effect of guttiferone E and carboplatin combination in lung tumor tissues. Our results demonstrate that after treatment with the guttiferone E and carboplatin combination, there was a significant decrease in the level of PARP (p<0.001), SIRT1 (p<0.001) and SIRT7 (p<0.001) proteins compared to the lysate from control group tumors (Figure 5A). PD-1 and its ligand PD-L1 are important in immunotherapy. In our study, we observed that there was also a significant decrease in the levels of PD-1 (p<0.01), PD-L1 (p<0.001), mTOR (p<0.001) and SOD2 (p<0.01) by administering guttiferone E and carboplatin combined as compared to the control (Figure 5C). These data suggest that the combination of guttiferone E and carboplatin has a synergistic effect in the regulation of apoptosis in the tumor tissue lysates compared to guttiferone E and carboplatin alone, through the modulation of immune cell-tumor cell checkpoint inhibition.
Effect of guttiferone E, carboplatin and their combination (Combo) on xenotransplants in athymic nude mice. A: Representative western blots and quantification of poly-ADP ribose polymerase (PARP), sirtuin 1 (SIRT 1), sirtuin 7 (SIRT 7) and β-actin in tumor tissue lysate. B: Representative western blots and quantification of programmed death-ligand 1 (PD-L1), programmed death 1 (PD-1), mechanistic target of rapamycin (mTOR), superoxide dismutase (SOD2), and β-actin in the tumor tissue lysate. The values are expressed as the mean±standard deviation (n=3). Statistical significance is indicated as: *p<0.05, **p<0.01, ***p<0.001 vs. the control, and ^p<0.05, ^^p<0.01 and ^^^p<0.001 vs. single treatments. Otherwise, there is no significant difference between the treatments.
Discussion
Cancer mortality and morbidity rates remain high regardless of all the efforts made in treatment. Multidrug resistance (MDR) is a major challenge to cancer treatment. More than 90% of cancer-related deaths are brought on by tumor metastasis and recurrence (52, 53). Intrinsic and extrinsic variables influence drug resistance in cancer cells, including genetic and epigenetic changes, drug efflux, DNA-repair mechanisms, apoptosis, and autophagy. There is currently no efficient anticancer treatment that can overcome MDR despite numerous types of compounds having been studied for their MDR-modifying function in vitro and in vivo (54, 55). In patients with cancer, phytochemicals may reduce the side-effects of traditional anticancer drugs by sensitizing the cancer cells to them, alone or in conjunction with another chemotherapeutic agent (56, 57). The NSCLC subtype, which is further divided into adenocarcinoma, squamous-cell carcinoma, and large-cell carcinoma, accounts for up to 85% of all incidences of lung cancer (58, 59). Surgery, targeted therapy using delivery systems or monoclonal antibodies, chemotherapy, radiation, immunotherapy, or a combination of these are all therapeutic modalities for NSCLC (60). The prognosis for patients with NSCLC is poor despite recent advancements in diagnostics and treatment methods. Hence, the key to finding efficient ways to enhance the prognosis of NSCLC is to identify and define possible molecular targets (61). The central objective of this study was to evaluate whether carboplatin and guttiferone E, alone or in combination, could overcome resistant lung cancer (with EGFR mutations) in tumor xenografts.
We conducted in-vitro studies in 2D and 3D cultures of osimertinib-resistant H1975 cells using guttiferone E, carboplatin, and their combination. Isobolographic studies conducted in vitro demonstrated that the combination showed synergistic behavior (combination index <1). We observed that the cytotoxic effect of guttiferone E was at a much lower molar concentration than carboplatin in both 2D and 3D assays. These results are not surprising because, in our laboratory, we have shown that cannabinoids, cannabidiol, and tetrahydrocannabivarin showed 4- to 5-fold lower IC50s as compared to doxorubicin against resistant MDA-MB231 breast cancer cells (47). This has also been demonstrated by other workers who used cannabidiol for other resistant tumors (62, 63).
The synergistic behavior of the combination was further investigated in an in-vivo osimertinib-resistant H1975 xenograft model, which showed that the combination showed significant tumor reduction (p<0.001) compared to the control. To understand the mechanism, western blot studies were conducted. Our western blot studies showed the combination significantly downregulated PARP (p<0.001), SIRT1 (p<0.001) and SIRT7 (p<0.001) proteins as compared to the control group. Further, there was also a significant decrease in the levels of PD-1 (p<0.01), PD-L1 (p<0.001), mTOR (p<0.001), and SOD2 (p<0.01).
SIRT7 has been linked to several human malignancies. Studies have shown that the levels of SIRT7 were elevated in human lung cancer tumor tissues (61, 64). We observed that levels of SIRT7 were elevated in the control group and were reduced in the group treated with the guttiferone E and carboplatin combination. Another study conducted with guttiferone G demonstrated that it substantially inhibited recombinant human SIRT1 and SIRT2 at micromolar concentrations (65).
SIRT1 is a protein deacetylase that requires nicotinamide adenine dinucleotide and is involved in several biological functions, including DNA-damage repair and the development of cancer (66-68). In chemoresistance-prone lung cancer cells, SIRT1 is increased. Genetic silencing or pharmacological inhibition of SIRT1 reversed chemoresistance by increasing DNA damage, activating apoptosis, and degrading X-ray repair cross-complementing group 1 (69). In our studies, we observed that the level of SIRT1 was increased in control animals while treatment with guttiferone E and carboplatin reduced the level of SIRT1 protein, which suggests its role in activating apoptosis.
SOD2 is a possible target for reducing tumor growth and inhibiting cancer cell invasion after radiation (70). The SOD2 level was increased in the control animals while treatment with guttiferone E and carboplatin combination significantly downregulated expression of this protein. Elevated levels of phospho-mTOR are observed in up to 90% and 60% of patients with lung adenocarcinoma and large-cell carcinoma, respectively, and lead to poor survival (71). In our study, we observed that the mTOR level was significantly reduced in the case of tumors treated with guttiferone E, carboplatin, and their combination.
The DNA-repair enzyme PARP was discovered as a therapeutic target in SCLC and verified in human and mouse models (72, 73). According to multiple studies, PARP1 inhibitors can be used in the clinic to treat or improve poor response to conventional cytotoxic chemotherapy in patients with NSCLC (74, 75). The PARP level was reduced with the guttiferone E and carboplatin combination, suggesting its role in lung cancer and induction of apoptosis as the possible pathway (76).
Tumors can activate various immune evasion pathways, including endogenous immune checkpoints. With the advent of drugs capable of targeting specific immune checkpoint mediators, such as cytotoxic T lymphocyte antigen-4, and, more recently, PD-1 and PD-L1, cancer immunotherapy has entered a transformative phase. These medications represent a significant advancement in oncological treatment, aiming to enhance the immune system’s ability to recognize and eliminate cancer cells effectively. This new approach has revolutionized cancer therapy by harnessing the body’s immune response to combat tumors, marking a paradigm shift towards more targeted and potentially curative treatment strategies. In various human cancer types, including lung, breast, and hematological malignancies, expression of PD-L1 has been linked to poor clinical outcomes. Research has suggested that regardless of histology, PD-L1 expression may range from 45% to 50% in tumor cells in NSCLC biopsies (77). Our study also showed that PD-1 and PD-L1 expression was high in the control tumor samples, while the combination treatment led to downregulation of both PD-1 and PD-L1 proteins (78). This suggests that our combination also works through immunotherapy of NSCLC as its novel mechanism.
One of the most often used platinum-based antitumor medications is carboplatin. It is specifically designed to treat cancers of the testicles, ovary, head, neck, and SCLC (33). DNA is the primary target of carboplatin, to which it binds effectively and inhibits transcription and replication to cause cell death (79). These DNA adducts influence several transduction pathways and result in tumor cell death or necrosis. We used carboplatin as a model drug in our studies because of its superior use against NSCLC tumors (expressing EGFR mutations) and it being used alternatively against tumors resistant to osimertinib.
Various guttiferones (F, K, and A) and their mechanisms of action have been well studied. Guttiferone F was shown to induce apoptosis by increasing the expression of cleaved pro-caspase-3 and PARP in PLC/PRF/5 human liver cancer cells and inhibited tumor growth in female SCID mice bearing subcutaneous xenografts of PLC/PRF/5 cells (80). Guttiferone A demonstrated cytotoxic, growth-inhibitory, and apoptosis-inducing effects with hepatic carcinoma (HepG2) cells, as well as preventative effects in mouse models of colorectal and tongue carcinogenesis (81). Guttiferone K successfully inhibited the motility and metastasis of hepatocellular carcinoma cells and restored abnormally low profilin 1 protein expression (46). In another study, combining guttiferone K with 5-fluorouracil reduced tumor size in mice and induced apoptosis and G0/G1 cell-cycle arrest in human colon HT-29 cells (82).
The effects of BRP on human monocytes and the prostate cell lines LNCaP and PC-3 have been studied. BRP demonstrated selective cytotoxic effects on prostate cancer cells and an immunomodulatory action (83), highlighting its potential for clinical trials with oncological patients and developing new immunomodulatory and antitumoral drugs. Some studies with guttiferone E have been reported, and in CCRF-CEM cells, guttiferone E substantially triggered apoptosis by activating caspases 3/7, caspase 8, and caspase 9, as well as by disrupting matrix metalloproteinase (26). Several natural products have been shown to overcome resistance to cancer. For example, epigallocatechin-3-gallate improved the chemotherapeutic efficacy of many anticancer drugs (such as doxorubicin) by blocking various drug transporters, lowering DNA methylation and histone acetylation, modulating the activity of miRNAs, and managing the tumor microenvironment (84).
Several combination therapies with natural products and carboplatin have been reported in the literature. In our laboratory, we have conducted studies with other natural products including cannabidiol and tetrahydrocannabivarin, and they were used in combination with doxorubicin in vitro and in vivo against doxorubicin-resistant MDA-MB231 tumor xenografts. The combination approach of cannabidiol and doxorubicin demonstrated the superior potential in tumor regression compared to control and single-agent treatments. It was found to be mediated by downregulation of transforming growth factor beta, specificity protein 1, NOD-like receptor family pyrin domain containing 3, p38 mitogen-activated protein kinase, and PD-L1. Further, cannabidiol/tetrahydrocannabivarin, combined with doxorubicin, inhibited H3K4 methylation and H2K5 acetylation, as demonstrated by western blotting and reverse transcription polymerase chain reaction (47). In another study, a combination of carboplatin and epigallocatechin-3-gallate induced cytotoxicity in esophageal cancer cells and induced apoptosis, leading to reduced cell proliferation (85). The gene expression ratio of caspases 8 and 9 was considerably increased in combined treatments at IC20 and IC25 of both drugs compared to monotherapies (p<0.05). Additionally, gallic acid increased the apoptotic effects of carboplatin and paclitaxel in human breast cancer MCF-7 cells, causing overexpression of P53 and BCL2-associated X, apoptosis regulator (86). Further, treatment with α-hederin (monodesmosidic triterpenoid saponin) in conjunction with carboplatin resulted in upregulation of caspase 3, cyclin D1, and induced phosphatidylinositol 3-kinase/protein kinase B/c-JUN N-terminal kinase protein phosphorylation (87). In another study, Rauwolfia vomitoria extract (Rau) was used alone and with carboplatin to increase its efficacy in ovarian cancer (88). In mouse xenograft studies, treatment with Rau alone showed significant suppression of tumor growth, achieving a 36% reduction at a low dose (20 mg/kg) and a more pronounced 66% reduction at a higher dose (50 mg/kg). This effect was comparable to the efficacy observed with carboplatin alone. Interestingly, combining Rau with carboplatin resulted in a synergistic enhancement of carboplatin’s effects, leading to a remarkable 90% reduction in tumor burden. This synergistic interaction underscores the potential of combining Rau and carboplatin as a potent therapeutic strategy for effectively suppressing tumor growth in experimental models.
To the best of our knowledge, this is the first study to report that guttiferone E and carboplatin combination has synergistic cytotoxic effects in both 2D and 3D cancer cells and led to regression of osimertinib-resistant H1975 tumor xenografts in a mouse model by reducing tumor volume, enhancing apoptosis and by immune checkpoint inhibition. Although we used only one resistant cell line, detailed investigation is undergoing with other lung cancer cell lines to understand the molecular mechanism of action of the combination of guttiferone E and carboplatin.
Conclusion
This groundbreaking study represents the inaugural exploration into the therapeutic potential of guttiferone E, a benzoyl phloroglucinol derivative sourced from BRP, specifically targeting drug-resistant lung cancer. The research assessed the effects of guttiferone E both independently and in conjunction with carboplatin, a platinum-based anticancer agent, in inhibiting the growth of osimertinib-resistant H1975 tumor xenografts. Results demonstrated a significant inhibition (p<0.05) of tumor growth when guttiferone E was combined with carboplatin, highlighting a synergistic effect between these compounds. The mechanism underlying the antitumor activity of guttiferone E was found to involve the induction of apoptosis, notably through the modulation of key proteins, such as SIRT1, SIRT7, and PARP. Additionally, guttiferone E exhibited actions on immune checkpoint pathways, particularly PD-1/PD-L1, suggesting its potential in enhancing immune-mediated antitumor responses. One of the study’s intriguing findings was the comparable efficacy of carboplatin and guttiferone E in promoting tumor regression at doses of 25 mg/kg and 10 mg/kg, respectively. This parity underscores the promising role of guttiferone E as a viable alternative to conventional chemotherapy approaches. Ongoing investigations are extending into various other tumor types including breast, colon, and gliomas, aiming to further elucidate and validate the broader therapeutic applicability of this natural compound in oncology.
Footnotes
Authors’ Contributions
AN, IK, MHT, JAAM, AMDM, AR and SD designed and performed the study, analyzed the results and wrote the article. MSS and JKB conceptualized the study, wrote and revised the article.
Conflicts of Interest
The Authors declare no competing interests.
Funding
This study was funded by several organizations. The National Institute on Minority Health and Health Disparities of the National Institutes of Health supported this research under Grant/Award Number U54 MD007582. The Consortium for Medical Marijuana Clinical Outcomes Research provided funding under Reference Award Number SUB00002097. Additionally, financial support was received from the São Paulo Research Foundation (FAPESP) under grant number 2017/04138-8. These funding sources enabled the execution of the study, contributing to its successful completion and findings.
- Received June 19, 2024.
- Revision received August 15, 2024.
- Accepted September 4, 2024.
- Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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