Research ArticleExperimental Studies

Intravenous Administration of Dehydroxymethylepoxyquinomicin With Polymer Enhances the Inhibition of Pancreatic Carcinoma Growth in Mice

HIROTO FUJISAKI, YUTAKA NAKANO, SACHIKO MATSUDA, KEIICHI SUZUKI, OSAMU ITANO, MASAYUKI TANAKA, SHUTARO HORI, YASUSHI HASEGAWA, YUTA ABE, HIROSHI YAGI, MINORU KITAGO, TOMOHIRO KONNO, KAZUHIKO ISHIHARA, KEIKO OHNO, SATOSHI KISHINO, KAZUO UMEZAWA and YUKO KITAGAWA
Anticancer Research December 2021, 41 (12) 6003-6012; DOI: https://doi.org/10.21873/anticanres.15419

Abstract

Background/Aim: Pancreatic cancer, which exhibits resistance to cytotoxic and molecular targeted drugs, has an extremely poor prognosis. Nuclear factor-κB (NF-κB) is constitutively activated in many pancreatic cancer cases. Although the NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) has exhibited anti-cancer effects in pancreatic cancer models, its poor solubility limits its use to intraperitoneal administration. Materials and Methods: Poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) forms stable polymer aggregates with DHMEQ. The stability of DHMEQ aggregated with PMB in the human blood was measured by high-performance liquid chromatography–mass spectrometry (HPLC-MS) ex vivo. Anti-pancreatic cancer effects in AsPC-1 and MIA PaCa-2 pancreatic cancer cells were evaluated by cell growth inhibition assay in vitro and tumor growth inhibition assay in vivo. Results: DHMEQ aggregated with PMB (PMB-DHMEQ) remained detectable after 60 min of incubation in the human blood, whereas DHMEQ aggregated with carboxymethyl cellulose (CMC-DHMEQ) was barely detectable. PMB–DHMEQ significantly inhibited AsPC-1 and MIA PaCa-2 cell growth in vitro compared to CMC–DHMEQ. Intravenous administration of PMB–DHMEQ reduced the tumor volume and liver metastasis compared to untreated or CMC–DHMEQ-treated mice. Conclusion: Aggregation with PMB improved the solubility of DHMEQ, and effectively inhibited pancreatic cancer cell growth both in vitro and in vivo.

Key Words:

Nuclear factor-κB (NF-κB) comprises a family of pleiotropic transcription factors that orchestrate the expression of numerous genes with key roles in growth, oncogenesis, differentiation, apoptosis, tumorigenesis, and immune and inflammatory responses (1, 2). NF-κB is a heterodimer mainly consisting of p65 (Rel A) and p50 proteins. Inhibitor of κB (IκB) binds to NF-κB dimers in unstimulated cells, masking their nuclear localization signals and thus maintaining them in an inactive state in the cytoplasm. NF-κB is then activated by the signal-induced degradation of IκB proteins, resulting in translocation of the NF-κB complex to the nucleus, in which it can activate the expression of specific genes encoding proteins such as the inflammatory mediators interleukin (IL)-1, IL-2, IL-6, IL-8, and tumor necrosis factor (TNF)-α; the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin; inhibitors of apoptosis proteins; and other anti-apoptotic proteins such as B-cell lymphoma-extra large and FLICE-like inhibitory protein (FLIP) (3-6).

Dehydroxymethylepoxyquinomicin (DHMEQ) is a specific NF-κB inhibitor that acts at the level of DNA binding in both canonical and non-canonical NF-κB–activating pathways. The mechanism by which DHMEQ inhibits NF-κB is well established. For example, NF-κB binding to DNA was induced by TNF-α and inhibited by DHMEQ in vitro as revealed via electrophoretic mobility shift assay (7, 8). In addition, DHMEQ inhibited nuclear localization of NF-κB in COS1 cells (7) and AsPC-1 pancreatic cancer cells (9). DHMEQ binds to a specific cysteine residue in p65, p50, RelB, and cRel via covalent binding (10), preventing their binding to DNA. DHMEQ thus decreased the expression of the anti-apoptotic proteins c-FLIP and survivin and increased that of the apoptotic proteins caspase-3 and caspase-8 (8).

Pancreatic adenocarcinoma has an extremely poor prognosis, with a 5-year survival rate of 10% (11, 12). It is highly resistant to cytotoxic and molecular targeted agents (13-15), and only 20-40% of patients are considered suitable candidates for surgical resection (16) because of the high incidence of metastasis or locally advanced disease at the time of diagnosis (17). The development of drugs for pancreatic cancer lags compared to other types of cancers, and new drugs are urgently needed to improve treatment options for these patients. Constitutive activation of NF-κB has been observed in approximately two-thirds of human pancreatic cancers, as well as in 90% of human pancreatic cell lines (18-20), indicating that NF-κB is a potential therapeutic target. A previous study revealed that proliferation of the human pancreatic cell line BxPC-3 was blocked by administration of the proteasome inhibitor PS-341, which indirectly blocks NF-κB activation (21). We previously reported that DHMEQ suppressed pancreatic cell carcinoma both in vitro and in vivo using subcutaneous (8), peritoneal (22) and liver metastasis models (9, 23). However, DHMEQ displays poor solubility, and it was therefore administered intraperitoneally in these previous trials. Its high degree of hydrophobicity means that specific measures are needed to allow its intravenous (IV) administration for clinical use.

We also previously reported the use of the amphiphilic phospholipid polymers poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)] (PMB) and poly(2-MPC-co-n-BMA-co-p-nitrophenylcarbonyloxyethyl methacrylate) (PMBN), which form stable polymer aggregates with DHMEQ in water and potentially enhance the solubility of hydrophobic substances (24). To improve the effect of DHMEQ for future clinical use, we examined the feasibility of IV injection of DHMEQ aggregated with PMB (PMB–DHMEQ) and compared the effects of the PMB formulation with those of a carboxymethyl cellulose (CMC) suspension (CMC-DHMEQ) on the growth of human pancreatic cell carcinoma cell lines AsPC-1 and MIA PaCa-2, which are known to express NF-κB (18-20), in vitro and in vivo using subcutaneous and liver metastasis mouse models.

Materials and Methods

Preparation of PMB-DHMEQ. Racemic DHMEQ (Figure 1A) was synthesized as described previously (25). PMB (Figure 1B) was constructed from 30 mol% MPC and 70 mol% BMA in monomer ratios, as described previously (24). The molecular weight of PMB was approximately 10 kDa. PMB–DHMEQ aggregates were prepared by dropping 1.5 ml of 1 mg/ml DHMEQ in dichloromethane into 3 ml of 15% PMB in phosphate-buffered saline (PBS), and the mixture was sonicated for 30 min at setting micro tip limit 2, 80% duty cycle using a Sonifer 450 (Branson Ultrasonics Corporation, Danbury, CT, USA) on ice, followed by mixing with a magnetic stirrer for 3 h at room temperature until all the dichloromethane had evaporated (PMB–DHMEQ) (Figure 1C). The final concentration of DHMEQ was 500 μg/ml. To prepare CMC–DHMEQ as a control, DHMEQ was suspended in 0.5% CMC at the same concentration of 500 μg/ml (Figure 1D).

Figure 1.
Figure 1.

Structural formula and appearance of dehydroxymethylepoxyquinomicin (DHMEQ). (A) Structural formula of racemic DHMEQ. (B) Structural formula of poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB). (C) Appearance of PMB–DHMEQ aggregates. (D) Appearance of carboxymethyl cellulose (CMC)–DHMEQ aggregates.

Measurement of complex size. The diameters of PMB, PMB–DHMEQ, CMC, and CMC–DHMEQ were measured using a NanoSight LM10 Nanoparticle Analysis System (NanoSight Ltd., Salisbury, UK).

Measurement of DHMEQ concentrations ex vivo. PMB–DHMEQ or CMC–DHMEQ at a concentration of 0.5 mg/ml was mixed with human whole blood, and DHMEQ concentrations were measured via high-performance liquid chromatography–mass spectrometry after 0, 5, 15, 30, and 60 min after separation of plasma, according to a previous report (26). Each experiment was repeated twice under each condition.

Cell lines and transfection. The pancreatic cell lines AsPC-1 and MIA PaCa-2 were obtained from the American Tissue Type Culture Collection (Rockville, MD, USA). AsPC-1 cells were maintained in Roswell Park Memorial Institute 1640 medium, and MIA PaCa-2 cells were grown in Dulbecco’s modified Eagle’s medium. Both media were supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, USA), and cells were grown in a 5% CO2 humidified incubator at 37°C. AsPC-1 cells were transfected with pcDNA3/GL vectors (LUX, Edinburgh, UK) stably expressing Gaussia princeps luciferase to generate AsPC-1-Luc cells.

Cell growth. The effects of DHMEQ on AsPC-1 and MIA PaCa-2 cell growth were analyzed using colorimetric assays to quantify cleavage of the tetrazolium salt WST-8 by mitochondrial dehydrogenases in viable cells. The dye formed can be quantified spectrophotometrically, and its levels are directly correlated to the number of metabolically active cells in the culture. Briefly, 1×104 AsPC-1 or MIA PaCa-2 cells were pre-incubated overnight in 5% CO2 at 37°C in a 96-well microculture plate, followed by incubation with various concentrations of PMB–DHMEQ or CMC–DHMEQ for 72 h or 20 μg/ml PMB–DHMEQ or CMC–DHMEQ for 24, 48, and 72 h. Each condition was replicated in six different wells. After each incubation period, the cells were incubated for an additional hour at 37°C in 110 μl of medium containing 10 μl of Cell Counting Kit-8 reagent (Dojindo, Molecular Technologies Inc., Tokyo, Japan). The samples were shaken for 30 s, and the absorbance at 450 nm (630 nm as reference) was measured using a microplate spectrophotometer (Tecan, Männedorf, Switzerland).

Animals. All animal experiments were conducted according to Keio University’s institutional guidelines for the care and use of laboratory animals in research. Female BALB/c Slc-nu/nu mice were purchased from Oriental Yeast (Tokyo, Japan). Mice were housed in a temperature-, humidity-, and ventilation-controlled vivarium with a 12-h/12-h light/dark cycle under specific pathogen-free conditions at Keio University Experimental Animal Center and fed sterile food and water. The total numbers of 6-week-old mice used in each experiment are indicated in the figure legends. At the end of the experiments, mice were euthanized via an intraperitoneal (IP) injection of >3 mg of pentobarbital sodium in saline.

Evaluation of the effects of PMB–DHMEQ and CMC–DHMEQ in vivo. AsPC-1 cells (1×106 cells in 100 μl of PBS) were implanted subcutaneously in mice. When the tumors reached approximately 5 mm in diameter, mice were randomized and divided into four groups of four mice each. In three of the groups, mice were administered PMB–DHMEQ IV every 3 days, CMC–DHMEQ IP every 3 days, or CMC–DHMEQ IP every day at a dose of 12 mg/kg, as described previously (8). Untreated mice served as a control group. Tumor lengths and widths were measured by the same observer using sliding calipers, and the tumor volume was calculated as 0.4× longest diameter × width2. Body weight growth rate was calculated as followed: day15 body weight divided by day 0 body weight.

Liver metastasis model and measurement of luciferase in whole mouse livers. A luciferase expression vector (Gaussia luciferase pcDNA3) was stably transfected into AsPC-1 cells (AsPC-1-Luc) as described previously. After 1 week of acclimatization, mice underwent laparotomy involving a 2-cm horizontal incision under anesthesia with 3% isoflurane, as reported previously (23). The portal vein behind the pancreatic head was identified and exposed. Fifty microliters of AsPC-1-Luc cells (1×107 cells/ml in PBS) were injected through the portal vein using gel foam to avoid bleeding and tumor cell leakage from the injected site. Mice were divided randomly into four groups containing four mice each. In addition to a control group, mice were administered IV PMB–DHMEQ every 3 days, IP CMC–DHMEQ every 3 days, or IP CMC-DHMEQ every day at a dose of 12 mg/kg, as described previously (8). Each treatment was started 1 day after tumor injection. After 3 weeks, whole livers were removed and homogenized using an ULTRA-TURRAX workstation (IKA Japan K.K., Osaka, Japan) in 10 ml of 1× Renilla Luciferase Assay Lysis Buffer (Promega KK, Tokyo, Japan). Luciferase was measured using a Renilla Luciferase Assay System (Promega KK).

Statistical analysis. Values are presented as the mean±standard deviation. Differences in tumor volumes, proliferation, and luciferase quantification were analyzed using unpaired Student’s t-tests. p<0.05 was considered statistically significant. All statistical analyses were performed using Excel.

Results

Particle size of PMB–DHMEQ complexes. The mean diameters of PMB alone, PMB–DHMEQ, CMC alone, and CMC–DHMEQ were 191, 266, 500, and 647 nm, respectively (Table I). The percentages of these complexes with diameters >1,000 nm were 0%, 0%, 8.7%, and 13%, respectively. CMC–DHMEQ was a white turbid liquid (Figure 1D), whereas PMB–DHMEQ was transparent (Figure 1C).

View this table:
Table I.

Particle diameters of PMB, PMB–DHMEQ, CMC, and CMC–DHMEQ.

Stability of DHMEQ in blood ex vivo. PMB–DHMEQ and CMC–DHMEQ were mixed with human blood to evaluate the stabilities of DHMEQ in blood. The concentrations of DHMEQ decreased to 30% (1.98 μg/ml) for PMB–DHMEQ and 20% (0.82 μg/ml) for CMC–DHMEQ after 30 min (Figure 2). DHMEQ complexed with CMC was barely detectable after 60 min, but 20% of the initial amount remained in blood mixed with PMB–DHMEQ (1.27 μg/ml, Figure 2). PMB–DHMEQ was thus more stable than CMC–DHMEQ in blood.

Figure 2.
Figure 2.

Stability of dehydroxymethylepoxyquinomicin (DHMEQ) in human blood ex vivo. Concentrations of DHMEQ in human blood containing carboxymethyl cellulose (CMC–DHMEQ) (A) and poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB)–DHMEQ (B) were measured by high-performance liquid chromatography–mass spectrometry (HPLC-MS) at 5, 15, 30, and 60 min after mixing. Each experiment was repeated twice.

Cytotoxic effect of PMB-DHMEQ complexes in pancreatic adenocarcinoma cells. We evaluated the cytotoxic effects of the complexes in pancreatic AsPC-1 and MIA PaCa-2 cells using in vitro proliferation assays. PMB–DHMEQ inhibited cell growth in a concentration-dependent manner, and its effects were stronger than those of CMC–DHMEQ (Figure 3A and B). Compared to the findings in control cells, the rates of AsPC-1 cell growth on day 3 were 0.56-fold for 20 μg/ml CMC–DHMEQ and 0.26-fold for 20 μg/ml PMB–DHMEQ (Figure 3C), whereas the rates for MIA PaCa-2 cells were 042- and 0.25-fold, respectively (Figure 3D). The differences between CMC–DHMEQ and PMB–DHMEQ at day 3 were significant (p<0.05) in both cell lines.

Figure 3.
Figure 3.

Growth inhibition assay using pancreatic cell lines. Relative proliferation is presented. Cells were incubated with various concentrations of dehydroxymethylepoxyquinomicin (DHMEQ) complexed with carboxymethyl cellulose (CMC) or poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) as indicated for 72 h (A, B). Cell growth without DHMEQ was used as a negative control (NC). Cell growth on days 1, 2, and 3 with PMB–DHMEQ or CMC–DHMEQ at 20 μg/ml are shown in (C) and (D). AsPC-1 (A, C) and MIA PaCa-2 cells (B, D) were used in this experiment. Cell growth on day 0 was used as a reference. NC indicates cells without DHMEQ. *p<0.05, **p<0.05.

Effects of PMB-DHMEQ on tumor size in vivo. To determine whether PMB–DHMEQ more strongly inhibited tumor growth than CMC-DHMEQ in vivo, AsPC-1 subcutaneous xenograft model mice were administered PMB–DHMEQ IV every 3 days, IP CMC–DHMEQ every 3 days, or IP CMC-DHMEQ every day. On day 15, tumors were approximately 35% smaller in the IV PMB–DHMEQ group than in the control group (62.8±13.7 mm3 vs. 176.5±41.1 mm3, p<0.05, Figure 4). The body weight growth rate from day 0 to day 15 was 1.18 times higher compared to the control group, 1.19 times higher in the IP CMC–DHMEQ every day group and every 3 days group, and 1.06 times higher in the IV PMB–DHMEQ group.

Figure 4.
Figure 4.

Tumor size in the AsPC-1 subcutaneous xenograft model. When tumors reached 5 mm in diameter, mice were given intravenous (i.v.) poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB)–dehydroxymethylepoxyquinomicin (DHMEQ) every 3 days, intraperitoneal (i.p.) carboxymethyl cellulose (CMC)–DHMEQ every 3 days, or i.p. CMC–DHMEQ every day as indicated at a dose of 12 mg/kg. *p<0.05 between i.v. PMB–DHMEQ every 3 days, and IP CMC–DHMEQ every 3 days. N=4/group.

Effect of PMB-DHMEQ on tumor metastasis. We established an AsPC-1 cell line stably expressing luciferase and assessed metastasis by measuring luciferase activity in the whole liver to evaluate the number of AsPC-1 cells. Luciferase activity was significantly lower in the IV PMB–DHMEQ group than in the other groups on day 21 (p<0.05), including a reduction of 15% compared to the control group (Figure 5). By contrast, IP CMC–DHMEQ administration every day or every 3 days did not prevent liver metastasis in this model.

Figure 5.
Figure 5.

Effects of poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB)–dehydroxymethylepoxyquinomicin (DHMEQ) on liver metastasis. AsPC-1-Luc cells (5×105) were injected into BALB/c Slc-nu/nu mice via the portal vein to produce a liver metastasis model. Mice were given intravenous (i.v.) poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB)–dehydroxymethylepoxyquinomicin (DHMEQ) every 3 days, intraperitoneal (i.p.) carboxymethyl cellulose (CMC)–DHMEQ every 3 days, or i.p. CMC–DHMEQ every day as indicated at a dose of 12 mg/kg. Each treatment started 1 day after tumor injection and continued for 3 weeks. Luciferase activity was measured in whole liver homogenates to quantify the degree of metastasis. *p<0.05. N=4/group.

Discussion

In this study, we demonstrated that DHMEQ could be solubilized using PMB, thus allowing it to be administered via IV injection, and the complexes exerted effects on cancer growth both in vitro and in vivo. IV therapy has advantages in terms of systemic administration for clinical use, and the IV route is a more common mode of administration than the IP route for cancer therapies. To the best of our knowledge, this study provided the first evidence of an anti-cancer effect of IV DHMEQ, a finding with implications for the clinical use of this compound, especially in pancreatic cancer, which generally has an extremely poor prognosis.

The mechanisms responsible for the increased anti-tumor effect of PMB–DHMEQ in this study remain unknown, and this represents a limitation of this study. However, the size of PMB–DHMEQ (mean 268 nm) was similar to that of verteporfin [255 nm for PMBN-verteporfin Ab (−)] (27), which may be associated with improved penetration into tumor cells as a result of enhanced permeability and retention (28). The high cellular affinity of PMB means that it is likely to increase both the solubility of DHMEQ and its penetration into cells (29), potentially making it an ideal solubilizing agent for DHMEQ. Moreover, DHMEQ was more stable in complex with PMB than in complex with CMC in blood ex vivo, suggesting that PMB may improve the stability of DHMEQ. DHMEQ has been revealed to penetrate RAW264.7 (murine macrophage-like cells) and MDA-MB-231 (human breast cancer cells) cells within 15 min and to inhibit NF-κB for more than 8 h in vitro (30), and a small increase in its stability may thus affect tumor growth in vivo.

We previously demonstrated the effectiveness of CMC–DHMEQ in a subcutaneous xenograft model using the PK-8 pancreas cell line (8) and an AsPC-1 cell liver metastasis model. We also compared CMC–DHMEQ alone with the combination of CMC–DHMEQ and gemcitabine (9). However, the current study was the first to compare IP CMC and IV PMB and confirm the greater efficacy of IV PMB–DHMEQ both in vitro and in vivo. The apparent efficacy of PMB–DHMEQ against pancreatic cancer was particularly promising given that drug delivery to pancreatic tumors is especially difficult because of their hypovascularity and poor perfusion (31, 32).

Until now DHMEQ was mostly injected into the peritoneal cavity to show anticancer (33) and anti-inflammatory (34) effects on various animal disease models. In these cases, DHMEQ is likely to act only in the peritoneal cavity, since DHMEQ does not appear in the blood. Therefore, we suggested the importance of NF-κB in peritoneal inflammatory cells on cancer progression and inflammation (33-35). On the other hand, DHMEQ is likely to reach cancer cells via the blood stream in case of intravenous administration of PMB-DHMEQ. If DHMEQ reaches to the cancer cells, it is possible to induce apoptosis and introduce anticancer activity. In fact, DHMEQ was shown to induce apoptosis in prostate carcinoma cells (36), thyroid carcinoma cells (37), and adult T-cell leukemia cells (38).

We used subcutaneous xenograft and portal vein metastasis models to assess cancer growth and metastasis. However, further studies employing spontaneous cancer and metastasis models would be valuable for establishing the potential clinical efficacy of DHMEQ in terms of the hypovascular and poorly perfused nature of pancreatic cancer. Furthermore, although the doses used showed positive anti-tumor effects, we did not evaluate the effects of long-term treatment with PMB or CMC, and the acute and chronic toxicities of PMB need to be investigated prior to its clinical application. Last, although we determined the stabilities of PMB–DHMEQ and CMC–DHMEQ in blood, further studies are needed to evaluate their in vivo stabilities.

In conclusion, the results of this study confirmed the feasibility of administering PMB–DHMEQ via IV injection and demonstrated its potential efficacy in the treatment of pancreatic cancer.

Acknowledgements

The Authors wish to thank Ms. Y. Nakamura and S. Matsuda for their continued technical support. We are grateful to the Collaborative Research Resources, School of Medicine, Keio University for technical support and reagents. We thank Joe Barber Jr., PhD, from Edanz (https://jp.edanz.com/ac), for editing a draft of this manuscript.

Footnotes

  • ↵‡ Current affiliation: Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi, Japan.

  • Authors’ Contributions

    HF, SM and KU conceived and designed the study. HF and SM performed all experiments, data curation, and drafted the manuscript. TK and KI synthesized PMB polymer and prepared PMB-DHMEQ aggregation. KO and SK designed measurement of DHMEQ in blood and performed experiments. KU synthesized DHMEQ and prepared PMB-DHMEQ aggregation. HF, YN, SM, KS, OI, MT, SH, YH, YA, HY, MK, and YK analyzed and interpretated the curated data. SM and KU supervised the project, and confirm the authenticity of all the raw data. All Authors revised the manuscript and approved the final version of the manuscript for publication.

  • Conflicts of Interest

    The co-author, YK received fundamental research funding from CHUGAI PHARMACEUTICAL CO., LTD. The sponsor had no control over the interpretation, writing, or publication of this work. The remaining Authors report no conflicts of interest.

  • Received October 7, 2021.
  • Revision received October 31, 2021.
  • Accepted November 4, 2021.

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Intravenous Administration of Dehydroxymethylepoxyquinomicin With Polymer Enhances the Inhibition of Pancreatic Carcinoma Growth in Mice
HIROTO FUJISAKI, YUTAKA NAKANO, SACHIKO MATSUDA, KEIICHI SUZUKI, OSAMU ITANO, MASAYUKI TANAKA, SHUTARO HORI, YASUSHI HASEGAWA, YUTA ABE, HIROSHI YAGI, MINORU KITAGO, TOMOHIRO KONNO, KAZUHIKO ISHIHARA, KEIKO OHNO, SATOSHI KISHINO, KAZUO UMEZAWA, YUKO KITAGAWA
Anticancer Research Dec 2021, 41 (12) 6003-6012; DOI: 10.21873/anticanres.15419
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