Research ArticleExperimental Studies

Aurelianolides from Aureliana fasciculata var. fasciculata Trigger Apoptosis With Caspase Activation in Human Leukemia Cells

GUSTAVO WERNECK DE SOUZA E SILVA, ANDRÉ MESQUITA MARQUES, ANA PAULA GREGÓRIO ALVES FONTÃO, SIMONE CRISTINA DE MOURA LIMA, MARIA AUXILIADORA COELHO KAPLAN, MARIA RAQUEL FIGUEIREDO and ANDRÉ LUIZ FRANCO SAMPAIO
Anticancer Research March 2023, 43 (3) 1245-1253; DOI: https://doi.org/10.21873/anticanres.16271

Abstract

Background/Aim: Aurelianolide A and B were identified and isolated from Aureliana fasciculata var. fasciculata leaves. Withanolides are naturally occurring C-28 steroidal lactone triterpenoids with cytotoxic and anticancer properties, among other relevant pharmacological activities. Herein we have described, for the first time, the cytotoxic effects of aurelianolides on human cancer cells. Materials and Methods: Aurelianolide A and B were tested on human leukemia cell lines: THP-1, MOLT-4, Jurkat, K562 and K562-Lucena 1. Results: For aurelianolide A, MOLT-4 had the lower IC50 (1.17 μM) and for aurelianolide B, Jurkat was the most susceptible cell line (IC50 2.25 μM). On the other hand, the multidrug resistant (MDR) cell line K562-Lucena 1 showed higher IC50 for both aurelianolides. Using 293T, a non-tumor embryonic kidney cell line, we observed an excellent selectivity index for both aurelianolides, from 2.24 (aurelianolide B in K562-Lucena 1) to 45.5 (aurelianolide A in MOLT-4). Aurelianolide A and B activated caspase 3/7 with consequent induction of apoptosis on Jurkat and K562-Lucena 1 cell lines. We have not observed induction of necrosis. Conclusion: Aurelianolides A and B have important cytotoxic effects on human leukemia cell lines by the activation of the caspase pathway.

Key Words:

Cancer is considered a multifactorial disease and one of the most significant public health challenges worldwide. Many genetic and environmental factors can increase the risk of developing cancer, compromising life expectancy in many countries (1). Drug resistance and severe side effects can limit the use of anticancer drugs and thus impair patients’ quality of life (2).

The pharmaceutical industry intensified the search for new drugs after the discovery of important bioactive substances based on natural occurring scaffolds (3). In comparison with synthetic drugs, plant derivatives tend to have more chemical diversity, including many chiral centers, more oxygen atoms, as well as varied ring systems (4). Due to this structural complexity, natural compounds tend to be more selective in relation to their targets than fully synthetic drugs, playing a prominent role in the development of new drugs, especially in oncology, where 53.3% of active components are considered of natural or of plant origin (5).

As part of the discovery strategies to search for new bioactive compounds from plant biodiversity, phytochemical and pharmacological studies have shown a series of promising metabolites with potential antitumor effects in in vitro tests (6). Pharmacological studies involving the class of withanolides metabolites, which are natural steroidal lactone triterpenoids, have suggested potential anticancer properties in human cancer cells in vitro, as well as tumor angiogenesis and metastasis suppression effects (7-9) via production of ROS, interference on cell cycle or cytoskeleton destabilization (10, 11). However, the molecular mechanism by which withanolides inhibit the proliferation of human cancer cells is still largely unknown.

The species Aureliana fasciculata var. fasciculata has been described as a new endemic species of the Solanaceae family in Brazil (12). The first studies with the species showed the presence of two new main withanolides named aurelianolide A and aurelianolide B (Figure 1), by Almeida-Lafetá et al. (13). Aurelianolides A and B can be considered as possible new leaders for planning the design of drugs with a focus on leishmaniasis and Trypanosoma cruzi (14, 15). Rare pharmacological investigations of these compounds contribute to a lack of understanding of the underlying mechanisms of action.

Figure 1.
Figure 1.

Structure of the Type 1 withanolide and the isolated compounds from Aureliana fasciculata var. fasciculata: Aurelianolide A and Aurelianolide B.

In this work, aurelianolide A and aurelianolide B, isolated from Aureliana fasciculata var. fasciculata are investigated for the first time from a pharmacological point of view, as promising leading compounds for the development of new anticancer drugs.

Materials and Methods

Reagents. If not stated otherwise, reagents and all best grade materials were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell lines. The THP-1 and MOLT-4 leukemia cell lines were purchased from Rio de Janeiro Cell Bank and Jurkat, K562, K562-Lucena 1 were kindly donated by Dr Juliana Echevarria (UFRJ; Brazil); the non-malignant epithelial-like cell line 293T was kindly donated by LATEB – Biomanguinhos/FIOCRUZ. All cell lines were maintained and cultivated in RPMI1640 medium supplemented with 10% FBS purchased from Gibco (Waltham, MA, USA), sodium pyruvate 1 mM, 100 U/I penicillin, 0.1 mg/ml streptomycin and 0.05 mg/ml gentamicin purchased from Sigma-Aldrich. Cells were used between passage 4 and 20.

Botanical material. The species Aureliana fasciculata (Vell.) Sendtner var. fasciculata was collected in the city of Simão Pereira, MG and was identified by botanist Dr. Rita de Cassia Almeida-Lafetá, deposited in RFA Herbarium (UFRJ, Rio de Janeiro, Brazil) under number 40829, and registered in the Brazilian Genetic Patrimony (SISGEN) under the number AB5D582.

Extract preparation and isolation of aurelianolides. For methanol extract, the fresh leaves were weighed, dried in an oven at 40°C for 48h and the extract was prepared according to Lima et al (14). The dried methanol extract was suspended in MeOH/H2O (3:7) and partitioned using an increasing order of polarity. The dichloromethane fraction was chromatographed on a Sephadex-LH 20 column using methanol/CHCl3 (3:1) as eluent, and the collected fractions were reunited and chromatographed on a preparative plate to obtain aurelianolide A and aurelianolide B, that were identified by 1H- and 13C-NMR spectroscopy, compared with the previously isolated compounds (13, 14).

ESI-MS analysis. Mass spectra were obtained from the high-resolution device in MicroTOFII Bruker electrospray ionization as described by Lima et al (14). The Aurelianolide A compound was obtained as white amorphous white solid with the molecular formula C30H40O8 as determined by HR-ESIMS [m/z 529.2807 (M+1)]. The Aurelianolide B compound was obtained as white crystals with the molecular formula C30H40O7 as determined by HR-ESIMS [m/z 513.2864 (M+1)].

NMR analysis. Structural determination of the isolated compounds was performed by nuclear magnetic resonance spectra of hydrogen (1H NMR) and carbon (13CNMR); these were obtained on a Varian device VNMRS-Gemini 500 spectrometer operating at a frequency of 400MHz/100MHz using CD3OD as solvent. COSY, HMBC and HSQC were performed and compared with the previously isolated compounds (13, 14).

Cellular cytotoxicity assay using the MTT colorimetric method. Twenty-four hours before treatment with the samples, 100 μl of the cell suspension (5×103 cells//well) were added to 96-well plates and maintained in a 5% CO2 atmosphere at 37°C. Treatment with aurelianolides was performed in multiple concentrations (from 0.01 to 100 μM), each in triplicate. After 48h, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) was added. After the incubation time (4h), plates were centrifuged (1,500 rpm for 10 min), the supernatant carefully aspirated, and formazan crystals were solubilized with DMSO (Sigma). Absorbance was measured at a wavelength of 540 nm in a microplate reader (Victor X5; Perkin Elmer, Waltham, MA, USA). The IC50 was calculated using GraphPad Prism version 5 (San Diego, CA, USA).

Selectivity index. 293T cells, a non-malignant embryonic kidney cell line, were cultured and treated with crescent concentrations of Aurelianolide A and B (from 0.01 to 100 μM) for IC50 determination and further selectivity index calculation using the formula:

Aurelianolide Selectivity Index=293T IC50 (μM)/Leukemia IC50 (μM)

Determination of caspase activity by flow cytometry analysis (FLICA). For caspase activity determination (16, 17), 105 cells were seeded (0.5 ml at 2×105 cells/ml) in a 24-well plate and incubated (24 h) in a 5% CO2 atmosphere at 37°C. Cells were treated in duplicate with aurelianolides, for 24 h. Negative controls consisted of cells incubated with the vehicle, while staurosporine (Sigma, 0.5 μM) was the positive control. Cells were stained according to the manufacturer’s instructions (Vybrant FAM Caspase-3 and -7 Assay Kit, Molecular Probes; Waltham, MA, USA). Experiments were performed three times in duplicate with a total of 104 events acquired. Data acquisition was performed with a FACScalibur flow cytometer and analysis using CellQuestTM software (Becton Dickinson; Franklin Lakes, NJ, USA). Forward and side scatters were set to exclude debris.

Determination of apoptosis/necrosis by flow cytometry analysis (Annexin V/propidium iodide). For apoptosis assays, 105 cells were seeded and treated as described for Caspase 3/7 assay and stained with Annexin V, as recommended by the manufacturer (Dead cell apoptosis kit, Molecular Probes). Propidium iodide (PI, Sigma) was added to the samples at the time of acquisition (within 5 min) to avoid false positives due to over-labeling with PI. Negative and positive controls consisted of cells incubated with vehicle and staurosporine (Sigma, 0.5 μM), respectively. Data acquisition was performed as described for the Caspase 3/7 assay. Apoptosis status was classified by the cell staining profile: early apoptosis (Annexin Vpos/PIneg); late apoptosis (Annexin Vpos/PIpos); necrosis (Annexin Vneg/PIpos).

Statistical analysis. Results were expressed as the mean±standard error of the mean (SEM). Statistical analysis was performed by one way ANOVA, followed by the Newman Keuls test, and considered significant when p<0.05 using GraphPad PRISM version 5 (San Diego, CA, USA).

Results

Effects of withanolides on leukemia cells. Aurelianolide A and aurelianolide B (0.01 to 100 μM) were tested on a panel of five human leukemia cell lines from different hematopoietic lineages. Cytotoxic effects of these isolated molecules were assessed for the first time, revealing that the aurelianolides A and B inhibit leukemic cell growth in a dose dependent manner (Figure 2) with low micromolar IC50 values in all of them (<10 μM; Table I).

Figure 2.
Figure 2.

Effects of the compounds aurelianolide A and aurelianolide B on leukemia cell proliferation. Cells were cultivated and treated with aurelianolide A (a) and aurelianolide B (b) in increasing concentrations (0.01 μM to 100 μM) for proliferation assessment using the MTT assay. Cells cultured in medium containing 0.25% DMSO were used as controls for 100% proliferation (dotted line). Points represent mean±standard error of mean (SEM) of triplicate experiments.

View this table:
Table I.

Selectivity index and IC50 of Aurelianolides on leukemia cells. Cells were cultivated and treated with aurelianolide A and B in increasing concentrations (0.01 μM to 100 μM) for cytotoxicity/proliferation assessment using MTT. The selectivity index was calculated as described in Materials and methods. Experiments were performed in triplicate and IC50 calculations were performed using Graph Pad Prism ver. 5.

Cell growth inhibition was observed in leukemia cell lines; aurelianolide A had lower IC50 in all of them, suggesting that this molecule is slightly more efficient than its counterpart, aurelianolide B. As shown in Figure 2, Jurkat cells were the most susceptible cell line, with the lowest IC50 values overall, with approximately 90% of growth inhibition starting at 10μM, whereas the K562-Lucena 1 cell line was the most resistant to treatment with the withanolides, even though the IC50 values obtained were fairly low (Table I). Previous findings have shown a resistant profile from K562-Lucena 1 cells, probably due to their MDR phenotype (18). The IC50 values for Jurkat and K562 Lucena-1 prompted us to choose these cell lines for further experiments to better understand the cytotoxicity triggered by aurelianolides A and B.

Selectivity of aurelianolide effects on leukemia cells. To evaluate the selectivity of aurelianolide effects on leukemia cells, we tested their cytotoxicity on 293T, a non-malignant epithelial-like cell line for selectivity index (SI) calculation. SI is the ratio of IC50 for non-malignant and IC50 for tumor cell lines. A higher SI suggests good selectivity toward tumoral cell lines. On average, we have observed high SI indexes for all the cell lines tested (Table I). Higher SI were obtained with aurelianolide A, with a mean of 24.24 (from 8.03 for K562 Lucena-1 to 45.50 for MOLT-4) while aurelianolide B had an average SI of 7.17 (from 2.24 for K562 Lucena-1 to 16.19 for Jurkat). Interestingly, K562 Lucena-1 showed low SI values while Jurkat displayed high values for both aurelianolides.

Caspase activity on leukemia cells after aurelianolides treatment. Caspases are well known for their key role in apoptosis activation and promotion. Herein, we show, for the first time, the pro-apoptotic activity of aurelianolides A and B on leukemia cells. Based on morphological analysis, we started the investigation of the effects of withanolides on caspase activity (Figure 3 and Figure 4). After 24 hours, both aurelianolides were able to activate caspases 3/7, with a strong activation with higher concentrations tested (2×IC50 equivalent). On Jurkat cells (Figure 3), the 2xIC50 equivalent treatment of aurelianolide A (3 μM) reached 69.20% of caspase 3/7 positive cells. Aurelianolide B was slightly less effective on triggering caspase activity, with 58.55% of caspase 3/7 positive cells at 5 μM (IC50).

Figure 3.
Figure 3.

Analysis of aurelianolide A- and B-induced apoptosis in Jurkat cells using flow cytometry. Cells were cultivated in medium (CTRL) or treated with aurelianolide A (1×IC50, left graph; 2×IC50 right graph), aurelianolide B (1×IC50, left graph; 2×IC50 right graph) or staurosporine (STAU; 0.5 μM) for 24h. Quadrants were determined based on unstained cells; numbers are percentages of a representative experiment. Apoptosis status was classified by the cell staining profile: early apoptosis (Annexin Vpos/PIneg; low right); late apoptosis (Annexin Vpos/PIpos; upper right); necrosis (Annexin Vneg/PIpos; upper left). Experiments were performed with a total of 104 events acquired. Data acquisition was performed with a FACScalibur flow cytometer and analysis was performed using CellQuestTM (Becton Dickinson, Franklin Lakes, NJ, USA).

Figure 4.
Figure 4.

Analysis of aurelianolide A- and B-induced apoptosis in K562-Lucena 1 cells using flow cytometry. Cells were cultivated in medium (CTRL) or treated with aurelianolide A (1×IC50, left graph; 2×IC50 right graph), aurelianolide B (1×IC50, left graph; 2×IC50 right graph) or staurosporine (STAU; 0.5 μM) for 24h. Quadrants were determined based on unstained cells; numbers are percentages of a representative experiment. Apoptosis status was classified by cell staining profile: early apoptosis (Annexin Vpos/PIneg; low right); late apoptosis (Annexin Vpos/PIpos; upper right); necrosis (Annexin Vneg/PIpos; upper left). Experiments were performed with a total of 104 events acquired. Data acquisition was performed with a FACScalibur flow cytometer and analysis using CellQuestTM (Becton Dickinson, Franklin Lakes, NJ, USA).

Aurelianolides A and B were equally effective at inducing caspase activity in K562-Lucena 1 cells (Figure 4), with greater efficiency of aurelianolide B in the concentrations used. At the higher dose (2×IC50 equivalent, 32 μM), 72.66% of caspase 3/7 positive cells were observed. Even though aurelianolide A was less efficient in activating caspases 3/7 in the concentrations used, it still showed a significant increase in the number of caspase 3/7 positive cells. These findings suggest, for the first time, an apoptotic induction potential of these newfound molecules.

Effects of aurelianolides on apoptosis of leukemia cells. Furthermore, based on the caspases 3/7 assay results we investigated the effects of aurelianolides on membrane annexin V expression to confirm the effects of these molecules on leukemia cell apoptosis (Figure 3 and Figure 4). At early apoptosis stages, phosphatidylserine is externalized allowing the binding of labelled annexin V. Using annexin V and propidium iodide staining, cells undergoing early apoptosis are annexin Vpos and PIneg. After 24 hour treatments, both aurelianolides significantly increased the number of cells on early apoptosis. Approximately 50% of Jurkat cells were apoptotic at this stage (Figure 5b) and 30% of the MDR K562-Lucena 1 cells (Figure 6b), at the 2×IC50 treatment. Using the combination of annexin V and propidium iodide (PI), we can detect different apoptotic stages by the cell staining profile: early apoptosis (Annexin Vpos/PIneg), late apoptosis (Annexin Vpos/PIpos), and necrosis (Annexin Vneg/PIpos). We observed that apoptosis levels increased considerably when cells were treated for 24 hours with aurelianolides. Aurelianolide A treatment increased late apoptosis numbers up to 22% in Jurkat cells and 15% in K562-Lucena 1 cells, whereas aurelianolide B increased late apoptosis levels up to 14% in Jurkat cells (Figure 5c) and 25% in K562-Lucena 1 cells (Figure 6c). A difference of cell response to treatment was observed; Aurelianolide B triggered a great increase in late apoptosis levels in K562-Lucena 1 cells while aurelianolide A triggered it on Jurkat cells.

Figure 5.
Figure 5.

Evaluation of caspase 3/7 activation (a) and annexin V/PI labelling (b, c, and d) on Jurkat cells. Cells were maintained in medium (CTRL) or treated with staurosporine (STAU; 0.5 μM) or aurelianolides (1×IC50 and 2×IC50). Samples were processed in duplicate and analyzed for caspase activation or in triplicate for Annexin V expression and PI labelling by flow cytometry. Apoptosis status was classified by the cell staining profile: early apoptosis (Annexin VPOS/PINEG); late apoptosis (Annexin VPOS/PIPOS); necrosis (Annexin VNEG/PIPOS). Columns represent mean±standard error of mean (SEM) of triplicate experiments. One-way ANOVA was performed, followed by the Newman Keuls test and differences were considered significant when p<0.05. *p<0.05 compared to control (CTRL) and +p<0.05 compared to 1×IC50.

Figure 6.
Figure 6.

Evaluation of caspase 3/7 activation (a) and annexin V/PI labelling (b, c, and d) on K562-Lucena 1 cells. Cells were maintained in medium (CTRL) or treated with staurosporine (STAU; 0.5 μM) or aurelianolides (1×IC50 and 2×IC50). Samples were processed in duplicate and analyzed for caspase activation or in triplicate for Annexin V expression and PI labelling by flow cytometry. Apoptosis status was classified by the cell staining profile: early apoptosis (Annexin VPOS/PINEG); late apoptosis (Annexin VPOS/PIPOS); necrosis (Annexin VNEG/PIPOS). Columns represent mean±standard error of mean (SEM) of triplicate experiments. One-way ANOVA was performed, followed by the Newman Keuls test and differences considered significant when p<0.05. *p<0.05 when compared to control (CTRL) and +p<0.05 when compared to 1×IC50.

Necrosis, as mentioned before, is characterized by annexin Vneg and PIpos cell staining. Necrosis is a tissue damaging type of cell death, with cellular contents being exteriorized without control. It is important for cancer therapy to find molecules that promote apoptotic pathways, instead of necrosis. In this regard, we have not observed necrosis after treatment with both aurelianolides (Figure 5d and Figure 6d).

Discussion

In this work, we have shown for the first time that two main withanolide metabolites, aurelianolides A and B, isolated from Aureliana fasciculata var. fasciculata have important antitumoral activity, triggering apoptosis with caspase activation. Aureliana fasciculata var. fasciculata is a native Brazilian species first described by Almeida-Lafetá et al. (13), belonging to the Solanaceae family. In the last decades, a great number of new withanolides have been isolated and characterized from several genera of Solanaceae family, showing a wide chemical diversity and significant pharmacological activities, such as immunoregulatory, antitumoral, anti-angiogenic, anti-invasive, and chemopreventive (19, 20). Recently, a withanolide rich fraction from Athanea velutina species (11) thought to contain aurelianolide A and aurelianolide B, in a mixture among other 10 withanolides, have shown a capability to be cytotoxic to murine melanoma cells B16F10. Herein, we have obtained micromolar IC50 values, for isolated aurelianolides A and B, on different human leukemia cells, unveiling the effects of these two compounds. On the other hand, until this date, no report has described the antitumoral potential of pure compounds isolated from Aureliana fasciculata var. fasciculata.

Withanolides isolated from Aureliana fasciculata var. fasciculata are classified as Type I, the most abundant form found in nature, endowed with the δ-lactone group. Its unmodified withanolide carbon frame shows the characteristics that are known to be related to the increase of antitumoral activity, which are the unsaturated ketone at ring A, an unsaturated δ-lactone group attached to C17, and the 5β,6β-epoxide that differs the two withanolides, which is present only in the aurelianolide A structure (21). The presence of this 5β,6β-epoxide has been shown to change the antitumoral effects of withanolides, reportedly increasing the cytotoxicity of the molecule (21). Concerning A. fasciculata aurelianolides, we have observed that aurelianolide A has a greater growth inhibition and decreased IC50 against every leukemia cell line, when compared to aurelianolide B. This increased cytotoxic activity is in accordance with the results of Lima and colleagues (14) that demonstrated an increased leishmanicidal activity of aurelianolide A, when compared to aurelianolide B, reinforcing our observation that the presence of a 5β,6β-epoxide is crucial for the biological activity of these compounds.

An anti-cancer drug should have a relatively high toxic concentration but with a very low active concentration. The selectivity index (SI) can be defined as the ratio of the toxic concentration of a sample against its effective bioactive concentration. For any anti-cancer activity of a sample, its cytotoxicity against non-malignant cell lines must be determined to calculate the SI value for the assessment of its viability for further studies or compound design. We have evaluated the cytotoxicity of aurelianolide A and B on 293T cells, a non-tumor embryonic kidney cell line, to determine the SI. Recently, Indrayanto and colleagues (22) suggested that samples with SI>10 are potential drug candidates and should be further investigated, and SI>3 suggest a prospective anti-cancer sample. We have observed that aurelianolide A and B in average have high SI values, from 4.02 (THP-1) to 45.5 (MOLT-4), with a SI<3 for K562 Lucena-1 (2.24), that could be associated with the MDR nature of this cell line that also has a higher IC50 for both aurelianolides. The SI for aurelianolide A, on average, is high (SI>10), while the SI for aurelianolide B was, on average, 7.17, fitting the aurelianolides as prospective candidates for further drug development.

Apoptosis is controlled cell death, different from necrosis, that is a dysregulated phenomenon, with spillage of intracellular content to extracellular spaces causing tissue damage. In further studies, we have shown that pure isolated aurelianolides A and B were able to trigger caspase 3/7 activation, monitored by flow cytometry (16, 17). Caspases are the most common pathway that trigger apoptosis in a cell. Both caspase 3 and 7 activation play a major role in inducing controlled cell death mechanisms, such as DNA fragmentation and cell shrinking (23). Aurelianolides A and B were equally able to induce caspase activity in K562-Lucena 1 cells (Figure 6a), with greater efficiency of aurelianolide B in the concentrations used. At the higher dose (2×IC50 equivalent, 32 μM), 72.66% of caspase 3/7 positive cells were observed. Even though aurelianolide A was less efficient in activating the caspases 3/7 in the concentrations used, it still showed a significant increase in the number of caspase 3/7 positive cells. These findings suggest, for the first time, an apoptosis triggering potential of aurelianolides. Our results with Aureliana fasciculata aurelianolides are in line with previous studies with other withanolides, on Jurkat cells, showing that these molecules have apoptosis-inducing and caspase activation activities (24, 25). The activation of caspase 3/7, plus the apoptosis levels observed, support the triggering and induction of apoptosis by these molecules, which can be a promising starting point for taking these compounds in an antitumoral drug development program.

Conclusion

Aurelianolides recently isolated from Aureliana fasciculata var. fasciculata have promising pharmacological activities with a very significant cytotoxic effect on a panel of leukemic cells, being original structures with several side chain modifications when compared to other withanolides (21). Aurelianolide A and B showed great cytotoxicity on Jurkat and K562-Lucena 1 leukemia cells via caspase 3/7 pathway and apoptosis activation. These compounds can be of great use for understanding the mechanisms underpinning cytotoxic actions of withanolides on cancer cells and development of new antitumoral drugs.

Acknowledgements

The Authors would like to thank Dr. Rita de Cássia Almeida-Lafetá from Faculdade de Educação Tecnológica do Rio de Janeiro (FAETERJ-Rio), for her kind identification and provision of Aureliana fasciculata (Vell.) Sendtner var. fasciculata. This work was supported by the CNPq under Grant PROEP/CNPQ - FAR/FIOCRUZ 08/2017 proc 407849/2017-3 and CNPq PROEPFAR II Proc 440018/2022-6.

Footnotes

  • Authors’ Contributions

    Conception and design of the work: A.L.F. Sampaio. Data collection: G.W.S. Silva, A.M. Marques, A.P.G.A. Fontão, S.C. de Moura Lima, A.L.F. Sampaio. Analysis and interpretation of the data: G.W.S. Silva, A.M. Marques, A.P.G.A. Fontão, S.C. de Moura Lima, M.A.C. Kaplan, M.R. Figueiredo, A.L.F. Sampaio. Statistical analysis: G.W.S. Silva, A.M. Marques, A.P.G.A. Fontão, S.C. de Moura Lima, M.A.C. Kaplan, M.R. Figueiredo, A.L.F. Sampaio. Drafting the manuscript: G.W.S. Silva, A.M. Marques, A.P.G.A. Fontão, S.C. de Moura Lima, M.A.C. Kaplan, M.R. Figueiredo, A.L.F. Sampaio. Critical revision of the manuscript: A.M. Marques, M.A.C. Kaplan, M.R. Figueiredo, A.L.F. Sampaio.

  • Conflicts of Interest

    The Authors declare no conflicts of interest.

  • Received November 16, 2022.
  • Revision received December 8, 2022.
  • Accepted December 9, 2022.

References

  1. ↵
    1. Bray F,
    2. Ferlay J,
    3. Soerjomataram I,
    4. Siegel RL,
    5. Torre LA and
    6. Jemal A
    : Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68(6): 394-424, 2018. PMID: 30207593. DOI: 10.3322/caac.21492
    OpenUrlCrossRefPubMed
  2. ↵
    1. Rayan A,
    2. Raiyn J and
    3. Falah M
    : Nature is the best source of anticancer drugs: Indexing natural products for their anticancer bioactivity. PLoS One 12(11): e0187925, 2017. PMID: 29121120. DOI: 10.1371/journal.pone.0187925
    OpenUrlCrossRefPubMed
  3. ↵
    1. Newman DJ and
    2. Cragg GM
    : Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. J Nat Prod 83(3): 770-803, 2020. PMID: 32162523. DOI: 10.1021/acs.jnatprod.9b01285
    OpenUrlCrossRefPubMed
  4. ↵
    1. Basmadjian C,
    2. Zhao Q,
    3. Bentouhami E,
    4. Djehal A,
    5. Nebigil CG,
    6. Johnson RA,
    7. Serova M,
    8. de Gramont A,
    9. Faivre S,
    10. Raymond E and
    11. Désaubry LG
    : Cancer wars: natural products strike back. Front Chem 2: 20, 2014. PMID: 24822174. DOI: 10.3389/fchem.2014.00020
    OpenUrlCrossRefPubMed
  5. ↵
    1. Abram M,
    2. Jakubiec M and
    3. Kamiński K
    : Chirality as an important factor for the development of new antiepileptic drugs. ChemMedChem 14(20): 1744-1761, 2019. PMID: 31476107. DOI: 10.1002/cmdc.201900367
    OpenUrlCrossRefPubMed
  6. ↵
    1. Dhar N,
    2. Razdan S,
    3. Rana S,
    4. Bhat WW,
    5. Vishwakarma R and
    6. Lattoo SK
    : A decade of molecular understanding of withanolide biosynthesis and in vitro studies in Withania somnifera (L.) Dunal: Prospects and perspectives for pathway engineering. Front Plant Sci 6: 1031, 2015. PMID: 26640469. DOI: 10.3389/fpls.2015.01031
    OpenUrlCrossRefPubMed
  7. ↵
    1. Carrero DAS,
    2. Batista PHJ,
    3. Souza LGS,
    4. Pinto FCL,
    5. Vasconcelos MA,
    6. Teixeira EH,
    7. Canuto KM,
    8. Santiago GMP,
    9. Silveira ER and
    10. Pessoa ODL
    : Withanolides from leaves of Nicandra physalodes. J Braz Chem Soc 29(1): 11-16, 2018. DOI: 10.21577/0103-5053.20170105
    OpenUrlCrossRef
    1. Chen LX,
    2. He H and
    3. Qiu F
    : Natural withanolides: an overview. Nat Prod Rep 28(4): 705-740, 2011. PMID: 21344104. DOI: 10.1039/c0np00045k
    OpenUrlCrossRefPubMed
  9. ↵
    1. Chinembiri TN,
    2. du Plessis LH,
    3. Willers C,
    4. Davids LM,
    5. Gerber M and
    6. du Plessis J
    : In vitro anti-melanoma efficacy and selectivity of Withania somnifera. Rev Bras Farmacogn 32: 421-432, 2022. DOI: 10.1007/s43450-022-00259-5
    OpenUrlCrossRef
  10. ↵
    1. Lacombe J,
    2. Cretignier T,
    3. Meli L,
    4. Wijeratne EMK,
    5. Veuthey JL,
    6. Cuendet M,
    7. Gunatilaka AAL and
    8. Zenhausern F
    : Withanolide D enhances radiosensitivity of human cancer cells by inhibiting DNA damage non-homologous end joining repair pathway. Front Oncol 9: 1468, 2020. PMID: 31970089. DOI: 10.3389/fonc.2019.01468
    OpenUrlCrossRefPubMed
  11. ↵
    1. Almeida AA,
    2. Lima GDA,
    3. Eiterer M,
    4. Rodrigues LA,
    5. A do Vale JA,
    6. Zanatta AC,
    7. Bressan GC,
    8. de Oliveira LL and
    9. Leite JPV
    : A Withanolide-rich fraction of Athenaea velutina induces apoptosis and cell cycle arrest in melanoma B16F10 cells. Planta Med 88(6): 429-439, 2022. PMID: 33853120. DOI: 10.1055/a-1395-9046
    OpenUrlCrossRefPubMed
  12. ↵
    1. Stehmann JR,
    2. Mentz LA,
    3. Agra M,
    4. Vignoli-Silva M,
    5. Giacomin L and
    6. Rodrigues IMC
    : Solanaceae in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro, 2015. Available at: http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB21134 [Last accessed on October 11, 2022]
  13. ↵
    1. Almeida-Lafetá RC,
    2. Ferreira JPF,
    3. Emerenciano VP and
    4. Kaplan MAC
    : Withanolides from Aureliana fasciculata var. fasciculata. Helv Chim Acta 93: 2478-2487, 2010. DOI: 10.1002/hlca.201000126
    OpenUrlCrossRef
  14. ↵
    1. Lima SCM,
    2. Pacheco JDS,
    3. Marques AM,
    4. Veltri ERP,
    5. Almeida-Lafetá RC,
    6. Figueiredo MR,
    7. Kaplan MAC and
    8. Torres-Santos EC
    : Leishmanicidal activity of Withanolides from Aureliana fasciculata var. fasciculata. Molecules 23(12): 3160, 2018. PMID: 30513673. DOI: 10.3390/molecules23123160
    OpenUrlCrossRefPubMed
  15. ↵
    1. Peres RB,
    2. Fiuza LFA,
    3. da Silva PB,
    4. Batista MM,
    5. Camillo FDC,
    6. Marques AM,
    7. de C Brito L,
    8. Figueiredo MR and
    9. Soeiro MNC
    : In vitro phenotypic activity and in silico analysis of natural products from Brazilian biodiversity on Trypanosoma cruzi. Molecules 26(18): 5676, 2021. PMID: 34577145. DOI: 10.3390/molecules26185676
    OpenUrlCrossRefPubMed
  16. ↵
    1. Darzynkiewicz Z,
    2. Pozarowski P,
    3. Lee BW and
    4. Johnson GL
    : Fluorochrome-labeled inhibitors of caspases: convenient in vitro and in vivo markers of apoptotic cells for cytometric analysis. Methods Mol Biol 682: 103-114, 2011. PMID: 21057924. DOI: 10.1007/978-1-60327-409-8_9
    OpenUrlCrossRefPubMed
  17. ↵
    1. Telford WG
    : Multiparametric analysis of apoptosis by flow cytometry. Methods Mol Biol 1678: 167-202, 2018. PMID: 29071681. DOI: 10.1007/978-1-4939-7346-0_10
    OpenUrlCrossRefPubMed
  18. ↵
    1. Maia RC,
    2. Wagner K,
    3. Cabral RH and
    4. Rumjanek VM
    : Heparin reverses Rhodamine 123 extrusion by multidrug resistant cells. Cancer Lett 106(1): 101-108, 1996. PMID: 8827053. DOI: 10.1016/0304-3835(96)04309-1
    OpenUrlCrossRefPubMed
  19. ↵
    1. Zhang H,
    2. Bazzill J,
    3. Gallagher RJ,
    4. Subramanian C,
    5. Grogan PT,
    6. Day VW,
    7. Kindscher K,
    8. Cohen MS and
    9. Timmermann BN
    : Antiproliferative withanolides from Datura wrightii. J Nat Prod 76(3): 445-449, 2013. PMID: 23252848. DOI: 10.1021/np300766p
    OpenUrlCrossRefPubMed
  20. ↵
    1. Wang HC,
    2. Tsai YL,
    3. Wu YC,
    4. Chang FR,
    5. Liu MH,
    6. Chen WY and
    7. Wu CC
    : Withanolides-induced breast cancer cell death is correlated with their ability to inhibit heat protein 90. PLoS One 7(5): e37764, 2012. PMID: 22701533. DOI: 10.1371/journal.pone.0037764
    OpenUrlCrossRefPubMed
  21. ↵
    1. Hussain H,
    2. Csuk R,
    3. Green IR,
    4. Ur Rehman N,
    5. Abbas G and
    6. Hussain W
    : Journey describing the cytotoxic potential of withanolides: a patent review. Recent Pat Anticancer Drug Discov 13(4): 411-421, 2018. PMID: 30091418. DOI: 10.2174/1574892813666180808154928
    OpenUrlCrossRefPubMed
  22. ↵
    1. Indrayanto G,
    2. Putra GS and
    3. Suhud F
    : Validation of in-vitro bioassay methods: Application in herbal drug research. Profiles Drug Subst Excip Relat Methodol 46: 273-307, 2021. PMID: 33461699. DOI: 10.1016/bs.podrm.2020.07.005
    OpenUrlCrossRefPubMed
  23. ↵
    1. Roberts JZ,
    2. Crawford N and
    3. Longley DB
    : The role of ubiquitination in apoptosis and necroptosis. Cell Death Differ 29(2): 272-284, 2022. PMID: 34912054. DOI: 10.1038/s41418-021-00922-9
    OpenUrlCrossRefPubMed
  24. ↵
    1. Oh JH,
    2. Lee TJ,
    3. Kim SH,
    4. Choi YH,
    5. Lee SH,
    6. Lee JM,
    7. Kim YH,
    8. Park JW and
    9. Kwon TK
    : Induction of apoptosis by withaferin A in human leukemia U937 cells through down-regulation of Akt phosphorylation. Apoptosis 13(12): 1494-1504, 2008. PMID: 19002588. DOI: 10.1007/s10495-008-0273-y
    OpenUrlCrossRefPubMed
  25. ↵
    1. Okamoto S,
    2. Tsujioka T,
    3. Suemori S,
    4. Kida J,
    5. Kondo T,
    6. Tohyama Y and
    7. Tohyama K
    : Withaferin A suppresses the growth of myelodysplasia and leukemia cell lines by inhibiting cell cycle progression. Cancer Sci 107(9): 1302-1314, 2016. PMID: 27311589. DOI: 10.1111/cas.12988
    OpenUrlCrossRefPubMed
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Aurelianolides from Aureliana fasciculata var. fasciculata Trigger Apoptosis With Caspase Activation in Human Leukemia Cells
GUSTAVO WERNECK DE SOUZA E SILVA, ANDRÉ MESQUITA MARQUES, ANA PAULA GREGÓRIO ALVES FONTÃO, SIMONE CRISTINA DE MOURA LIMA, MARIA AUXILIADORA COELHO KAPLAN, MARIA RAQUEL FIGUEIREDO, ANDRÉ LUIZ FRANCO SAMPAIO
Anticancer Research Mar 2023, 43 (3) 1245-1253; DOI: 10.21873/anticanres.16271
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