Cucurbitacin D exhibits its anti-cancer effect in human breast cancer cells by inhibiting Stat3 and Akt signaling

Abstract

Cucurbitacins are triterpenoids commonly found in Cucurbitaceae and Cruciferae and have long been used in traditional medicine. Cucurbitacins demonstrate anti-inflammatory and anti-cancer activities. We investigated whether cucurbitacin D affects viability in breast cancer cells and its mechanism of action. An MTT assay was used to measure the viability of breast cancer cells. Western blot analysis was used to measure the expression of various modulators, such as p-p53, p-Stat3, p-Akt, and p-NF-κB. Doxorubicin and cucurbitacin D affected the viability of MCF7, MDA-MB-231, and SKBR3 cells. Cucurbitacin D and doxorubicin increased p-p53 expression in MCF7, SKBR3, and MDA-MB-231 cells. Cucurbitacin D suppressed p-Akt, p-NF-κB, and p-Stat3 expression in MCF7, MDA-MB-231, and SKBR3 cells. Doxorubicin alone did not decrease p-Akt and p-Stat3 levels. Cucurbitacin D decreased p-NF-κB and p-Stat3 levels. Doxorubicin in combination with cucurbitacin D increased p-p53 levels and suppressed Akt, NF-κB, Stat3, and Bcl-2 expression more than cucurbitacin D alone. Our results clearly demonstrate that cucurbitacin D could be a useful compound for treating human breast cancer.

Keywords

breast cancer cucurbitacin D doxorubicin MCF7 cells MDA-MB-231 cells SKBR3 cells

Background

Breast cancer is one of the most common cancers in women and is the second leading cause of cancer-related mortality among women. Approximately 1.3 million people worldwide suffer from this disease.^1,2 Many factors, such as genetic disorders, a western lifestyle, and dietary patterns, are considered as causes of breast cancer. Fortunately, there have been improvements in the survival period and quality of life with breast cancer therapy.^3,4

Doxorubicin is an anthracycline antibiotic that is a US Food and Drug Administration (FDA)-approved chemotherapeutic drug. It has been widely used for the treatment of several cancers. Doxorubicin therapy has been recommended as the first-line treatment in the 2016 National Comprehensive Cancer Network’s breast cancer guidelines.⁵ Therefore, doxorubicin is a very important agent used in breast cancer therapy. Doxorubicin induces DNA damage and leads to cancer cell apoptosis.^6–9 However, resistance to doxorubicin develops in nearly 50% of treated patients.¹⁰ Therefore, resistance to doxorubicin remains the major obstacle to successful treatment. The MCF7 cell line has an induced upregulation of activated Stat3 and shows resistance to doxorubicin.^11–13

Signal transducers and activators of transcription (Stat) proteins are transcription factors. The Stat family is composed of Stats 1, 2, 3, 4, 5a, 5b, and 6. Stat3 activation is related to tumor development, for example, cell proliferation, survival, invasion, angiogenesis, and metastasis.^14–18 Also, the Akt pathway has been shown to play a key role in several cancers.¹⁹ Akt activation is associated with breast cancer and poor clinical outcomes.²⁰

Recently, interest in the anti-cancer effects of natural products is increasing, and research is actively underway.^21–24 Cucurbitacins are triterpenoids commonly found in Cucurbitaceae and Cruciferae and have been used in traditional medicine for a long time.²⁵ Cucurbitacins demonstrate anti-inflammatory and anti-cancer activities.²⁶ Cucurbitacins have a high toxicity, but this is not a problem because the toxic dose is larger than the active dose. Cucurbitacins have potential as a new drug for inhibiting cancer progression.²⁷ There are several types of cucurbitacins. Cucurbitacin B induces cell cycle arrest by decreasing c-Myc in human breast cancer cells.²⁸ Cucurbitacin E inhibits proliferation in prostate cancer cells and causes disruption of the cytoskeleton structure.²⁹ Many studies have revealed that cucurbitacin D induces apoptosis by suppressing the activation of NF-κ B and Stat3.^11,30 Also, it is known that cucurbitacin D induces apoptosis and autophagy in human T cell leukemia cells.³¹

In this study, we investigated the anti-cancer effect of cucurbitacin D in several breast cancer cell lines. For this purpose, we examined whether cucurbitacin D affects viability in MCF7 (ER positive), SKBR3 (Her2 positive), and MDA-MB 231 (triple negative) cells and its mechanism of action.

Materials and methods

Reagents and antibodies

Cucurbitacin D was purchased from Extrasynthese (Genay, France). Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA). The antibodies against phospho-Akt, phospho-Stat3 (Tyr705), phospho-NF-κB p65 (Ser536), phospho-p53, total Akt, total Stat3, and total p53 were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibodies against actin, Bcl-2, phospho-Erk, total Erk, and total NF-κB were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The tubulin antibody was obtained from Sigma–Aldrich.

Cell culture

MCF7, SKBR3, and MDA-MB 231 breast cancer cells obtained from the American Type Culture Collection (ATCC) were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 100 U/mL antibiotics-antimycotics (Invitrogen). Cells were maintained at 37°C in a humidified incubator with 5% CO₂.

Cell viability assay

Cell viability was measured using the MTT assay. Cells were plated in 96-well flat bottom tissue culture plates at a density of 3 × 10³ cells/well and incubated for 24 h. Cells were cultured for an additional 24 h with cucurbitacin D (0.125–16 μg/mL) or doxorubicin (0.04–25 μM). After incubation, MTT reagents (0.5 mg/mL) were added to each well, and the plates were incubated in the dark at 37°C for another 2 h. The medium was removed, formazan was dissolved in DMSO, and the optical density was measured at 570 nm using an ELISA plate reader.

Western blot analysis

Cells were harvested, incubated in one volume of lysis buffer (50 mM Tris-Cl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, and protease inhibitor) for 20 min and centrifuged at 13,000 r/min and 4°C for 20 min. Aliquots containing 20 μg of protein were separated by SDS-polyacrylamide gel electrophoresis using 8%–12% gels and transferred to nitrocellulose membranes (Protran nitrocellulose membrane; Whatman, UK). Membranes were blocked with 5% nonfat milk and probed with specific primary antibodies. Membranes were then incubated with horseradish peroxidase-conjugated secondary IgG antibody (Calbiochem, San Diego, CA, USA) and visualized using an enhanced chemiluminescence detection system (Amersham ECL kit; Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA).

Statistical analysis

All experimental results are expressed as the mean ± standard deviation (SD) or mean ± SD of the mean of at least three separate tests. Student’s t-test was used for comparisons, and a P-value < 0.05 was considered statistically significant. All statistical analyses were performed using PRISM software (GraphPad Software, Inc., La Jolla, CA, USA).

Results

Effect of doxorubicin and cucurbitacin D on MCF7, SKBR3, and MDA-MB-231 cell viability

We investigated whether doxorubicin and cucurbitacin D affected the viability of MCF7, MDA-MB-231, and SKBR3 cells. For that purpose, all MCF7, MDA-MB-231, and SKBR3 cells were treated with different concentrations of doxorubicin (0.005, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, and 10 μM) and cucurbitacin D (0.0025, 0.025, 0.05, 0.25, 0.5, and 2.5 μg/mL) for 24 h. Cell viability was then measured by MTT assay. We found that doxorubicin significantly suppressed growth in a dose-dependent manner in MCF7, MDA-MB-231, and SKBR3 cells (Figure 1(a)). We also found that cucurbitacin D significantly suppressed growth in all MCF7, MDA-MB-231, and SKBR3 cells in a dose-dependent manner (Figure 1(b)). Our results indicated that cucurbitacin D and doxorubicin significantly suppressed growth in MCF7, MDA-MB-231, and SKBR3 cells.

Figure 1.

Effect of doxorubicin and cucurbitacin D on MCF7, SKBR3, and MDA-MB 231 cell viability. MCF7, SKBR3, and MDA-MB 231 cells were treated with different concentrations of doxorubicin and cucurbitacin D for 24 h. Cell viability was then measured using the MTT assay. Data are presented as the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 by Student t-test.

Cucurbitacin D and doxorubicin increased p53 expression in MCF7, SKBR3, and MDA-MB-231 cells

We investigated whether doxorubicin and cucurbitacin D increased p-p53 levels in MCF7, SKBR3, and MDA-MB-231 cells. p53 has different multiple biological functions because it can regulate the expression of over 100 genes. p53 is a tumor suppressor gene that induces apoptosis, cell cycle arrest, and DNA repair.³² For this experiment, we treated MCF7, SKBR3, and MDA-MB-231 cells with cucurbitacin D (0.5, 1, 2, and 4 μg/mL) or doxorubicin (0.25, 0.5, 1, and 2 μM) and performed western blot analyses. We found that cucurbitacin D and doxorubicin increased the levels of p-p53 in MCF7, SKBR3, and MDA-MB-231 cells (Figure 2(a)–(c)).

Figure 2.

Cucurbitacin D and doxorubicin increased p-p53 levels in MCF7, SKBR3, and MDA-MB-231 cells. (a) MCF7, (b) SKBR3, and (c) MDA-MB-231 cells were treated with different concentrations of doxorubicin and cucurbitacin D for 24 h. Whole-cell lysates were analyzed by western blot to measure p-p53.

Cucurbitacin D suppressed Akt, NF-κB, and Stat3 expression in MCF7, SKBR3, and MDA-MB-231 cells

We investigated whether doxorubicin and cucurbitacin D inhibit Akt and Stat3 signaling in MCF7, MDA-MB-231, and SKBR3 cells. Activated-Akt, activated-NF-kB, and phospho-Stat3 play a critical role in cancer cells by increasing migration and invasion. Thus, Akt, Stat3, and NF-κB expression levels are very important in cancer therapy.^33–35 For this experiment, we treated MCF7, MDA-MB-231, and SKBR3 cells with cucurbitacin D (0.5, 1, 2, and 4 μg/mL) or doxorubicin (0.25, 0.5, 1, and 2 μM) and performed western blot analyses. We found that cucurbitacin D decreased the levels of p-Akt and p-Stat3 in MCF7, MDA-MB-231, and SKBR3 cells. However, doxorubicin alone did not decrease p-Akt and p-Stat3 levels (Figure 3(a)). When we treated MCF7 cells with IL-6, we found that cucurbitacin D decreased p-NF-κB and p-Stat3 expression (Figure 3(b)).

Figure 3.

Cucurbitacin D suppressed the expression of p-Akt, p-NF-κB, and p-Stat3 in human breast cancer cells. Constitutive activation of Akt and Stat3 was detected in MCF7, SKBR3, and MDA-MB 231 cells. Doxorubicin increased constitutive Stat3 phosphorylation in a dose-dependent manner in MCF7, SKBR3, and MDA-MB 231 cells. (a) Cucurbitacin D decreased p-Akt and p-Stat3 expression in MCF7, SKBR3, and MDA-MB 231 cells. MCF7 cells were treated with cucurbitacin D (0.5 μg/mL) and IL-6 (10 ng/mL). IL-6 increased constitutive NF-κB and Stat3 phosphorylation in MCF7 cells. (b) Cucurbitacin D suppressed p-NF-κB and p-Stat3 expression in MCF7 cells. Whole-cell lysates were analyzed by western blot with anti-pAkt, anti-Akt, anti-pNF-κB, anti-NF-κB, anti-pStat3, anti-Stat3, anti-actin, and anti-tubulin antibodies.

Combined effect of doxorubicin and cucurbitacin D on MCF7, SKBR3, and MDA-MB-231 cell viability

We investigated whether the combination of doxorubicin and cucurbitacin D affected the viability of MCF7, MDA-MB-231, and SKBR3 cells. For that purpose, we treated MCF7, MDA-MB-231, and SKBR3 cells with cucurbitacin D (0.5, 1, and 2 μg/mL) in the presence of doxorubicin (1 μM). Cell viability data from MTT assays were then analyzed by CompuSyn software to test a synergistic effect.³⁶ A combination of cucurbitacin D with doxorubicin showed the synergism. We found that cucurbitacin D combined with doxorubicin significantly suppressed cell growth more than cucurbitacin D alone (Figure 4(a)). Doxorubicin (1 μM) alone did not induce characteristic cell death morphology, but cucurbitacin D (2 μg/mL) alone induced characteristic cell death morphology in MCF7 and MDA-MB-231 cells. Additionally, doxorubicin combined with cucurbitacin D induced characteristic cell death morphology (Figure 4(b)).

Figure 4.

Effects of combined treatment with cucurbitacin D and doxorubicin on morphology and viability in MCF7 and MDA-MB-231 cells. MCF7, SKBR3, and MDA-MB 231 cells were treated with different concentrations of doxorubicin, cucurbitacin D, or cucurbitacin D (0.5 and 2 μg/mL) and doxorubicin (1 μM) for 24 h. Combinational treatments of cucurbitacin D with doxorubicin. The effect is a cell viability and CI is combination index. (a) Synergistic effects were analyzed with CompuSyn software. Cell viability was then measured using the MTT assay. Indicated MCF7 and MDA-MB-231 cells were photographed using a phase-contrast microscope. (b) A change in cell morphology was observed in MCF7 and MDA-MB-231 cells (magnification ×20). Data are presented as the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 by Student t-test.

Combination of cucurbitacin D with doxorubicin increased p-p53 levels and suppressed Akt, NF-κB, Stat3, and Bcl-2 expression in MCF7 cells

We investigated whether doxorubicin combined with cucurbitacin D increased p-p53 levels and suppressed Akt, NF-κB, Stat3, and Bcl-2 expression in MCF7 cells. We found that cucurbitacin D combined with doxorubicin significantly increased p-p53 levels and suppressed p-Akt, p-NF-κB, p-Stat3, and Bcl-2 levels more than doxorubicin or cucurbitacin D alone (Figure 5(a) and (b)).

Figure 5.

Combined treatment with cucurbitacin D and doxorubicin suppressed p-Akt, p-Erk, p-NF-κB, and p-Stat3 expression in MCF7 cells. MCF7 cells were treated with cucurbitacin D (0.5 μg/mL) and doxorubicin (1 μM). (a) Cucurbitacin D (0.5 μg/mL) and doxorubicin (1 μM) decreased p-Akt, p-Erk, p-NFκB, and p-Stat3 expression in MCF7 cells. (b) Cucurbitacin D (0.5 μg/mL) and doxorubicin (1 μM) decreased Bcl-2 levels and increased p-p53 levels. Whole-cell lysates were analyzed by western blot with anti-pAkt, anti-Akt, anti-pErk, anti-Erk, anti-pNF-κB, anti-NF-κB, anti-pStat3, anti-Stat3, anti-Bcl-2, anti-p-p53, anti-p53, and anti-tubulin antibodies.

Discussion

In this study, we found that cucurbitacin D decreased cell proliferation by inhibiting Akt, NF-κB, and Stat3 signaling in the human breast cancer cells MCF7, MDA-MB-231, and SKBR3.

Trichosanthes kirilowii tuber extract is a traditional medicine used in East Asia for patients with diabetes symptoms.³⁷ Recently, it was reported that cucurbitacin D, isolated from Trichosanthes kirilowii tuber extract, induces apoptosis through inhibiting Stat3 activity in breast cancer cells.³⁰ In addition, cucurbitacin D is known to induce apoptosis and cell cycle arrest in ovarian, cervical, lung, and hepatocellular carcinoma.^38–41 Therefore, we investigated whether doxorubicin and cucurbitacin D affected the viability of MCF7, MDA-MB-231, and SKBR3 cells. We found that cucurbitacin D and doxorubicin significantly suppressed cell growth in MCF7, MDA-MB-231, and SKBR3 cells.

Cucurbitacin B induces apoptosis in colon cancer cells expressing p53⁴² and enhances the regulatory effect of p53-specific CTL in 16HBE/BPDE tumor cells by inhibiting JAK2/Stat3 activation.⁴³

Cucurbitacin E decreases the viability of pancreatic cancer cells by suppressing Stat3 phosphorylation and upregulating tumor suppressor p53.⁴⁴ We investigated whether doxorubicin and cucurbitacin D increased p-p53 levels in MCF7 and MDA-MB-231 cells. We found that cucurbitacin D and doxorubicin increased the levels of p-p53 in MCF7 and MDA-MB-231 cells.

Doxorubicin-treated MCF7 cells have shown to contain activated Stat3. Stat3 activation is a predictive marker of drug resistance because it is linked to the development of doxorubicin resistance in cancer cell lines.^11,45,46 We found that cucurbitacin D decreased the levels of p-Akt and p-Stat3 in MCF7, MDA-MB-231, and SKBR3 cells. However, doxorubicin alone did not decrease p-Akt and p-Stat3 levels. We treated MCF7 cells with IL-6 to confirm whether cucurbitacin D inhibits the activity of NF-κB and Stat3. We found that cucurbitacin D decreased p-NF-κB and p-Stat3 levels. We investigated whether doxorubicin combined with cucurbitacin D affected the viability of MCF7, MDA-MB-231, and SKBR3 cells. We found that cucurbitacin D combined with doxorubicin significantly suppressed cell growth more than cucurbitacin D alone. Moreover, doxorubicin combined with cucurbitacin D changed the characteristic cell death morphology. Cucurbitacin D combined with doxorubicin significantly increased p-p53 levels and suppressed p-Akt, p-NF-κB, p-Stat3, and Bcl-2 levels more than doxorubicin or cucurbitacin D alone. Targeting Akt, NF-κB, and Stat3 may also be useful for treating breast cancer. Our study clearly demonstrates that cucurbitacin D inhibited Akt, NF-κB, and Stat3 activity in breast cancer cells and that cucurbitacin D combined with doxorubicin significantly suppressed cell growth more than cucurbitacin D alone.

Conclusion

Our study clearly demonstrates that cucurbitacin D inhibited Akt, NF-κB, and Stat3 activity in breast cancer cells and that doxorubicin combined with cucurbitacin D increased p-p53 levels and suppressed Akt, NF-κB, Stat3, and Bcl-2 expression more than cucurbitacin D alone. These results clearly demonstrate that cucurbitacin D could be used as a compound for treating human breast cancer.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This research was supported by a grant from the Korean Medicine R&D Project of the Ministry of Health and Welfare (B110043).

References

Siegel

Naishadham

Jemal

(2012) Cancer statistics, 2012. CA: A Cancer Journal for Clinicians 62: 10–29.

Fan

Strasser-Weippl

et al . (2014) Breast cancer in China. The Lancet Oncology 15: e279–e289.

Torre

Bray

Siegel

et al . (2015) Global cancer statistics, 2012. CA: A Cancer Journal for Clinicians 65: 87–108.

Aggarwal

Sethi

Ahn

et al . (2006) Targeting signal-transducer-and-activator-of-transcription-3 for prevention and therapy of cancer: Modern target but ancient solution. Annals of the New York Academy of Sciences 1091: 151–169.

Gradishar

Salerno

(2016) NCCN guidelines update: Breast cancer. Journal of the National Comprehensive Cancer Network 14: 641–644.

Tacar

Sriamornsak

Dass

(2013) Doxorubicin: An update on anticancer molecular action, toxicity and novel drug delivery systems. Journal of Pharmacy and Pharmacology 65: 157–170.

Rabbani

Finn

Ausio

(2005) The anthracycline antibiotics: Antitumor drugs that alter chromatin structure. BioEssays 27: 50–56.

Szakacs

Paterson

Ludwig

et al . (2006) Targeting multidrug resistance in cancer. Nature Reviews Drug Discovery 5: 219–234.

Zheng

Kallio

Harkonen

(2007) Tamoxifen-induced rapid death of MCF-7 breast cancer cells is mediated via extracellularly signal-regulated kinase signaling and can be abrogated by estrogen. Endocrinology 148: 2764–2777.

10.

Taylor

Dalton

Parrish

et al . (1991) Different mechanisms of decreased drug accumulation in doxorubicin and mitoxantrone resistant variants of the MCF7 human breast cancer cell line. British Journal of Cancer 63: 923–929.

11.

Kim

Hong

et al . (2015) Cucurbitacin D induces cell cycle arrest and apoptosis by inhibiting STAT3 and NF-kappaB signaling in doxorubicin-resistant human breast carcinoma (MCF7/ADR) cells. Molecular and Cellular Biochemistry 409: 33–43.

12.

Wang

Kotamraju

Konorev

et al . (2002) Activation of nuclear factor-kappaB during doxorubicin-induced apoptosis in endothelial cells and myocytes is pro-apoptotic: The role of hydrogen peroxide. Biochemical Journal 367: 729–740.

13.

Gariboldi

Ravizza

Molteni

et al . (2007) Inhibition of Stat3 increases doxorubicin sensitivity in a human metastatic breast cancer cell line. Cancer Letters 258: 181–188.

14.

Bromberg

Wang

(2009) Inflammation and cancer: IL-6 and STAT3 complete the link. Cancer Cell 15: 79–80.

15.

Kamran

Patil

Gude

(2013) Role of STAT3 in cancer metastasis and translational advances. BioMed Research International 2013: 421821.

16.

Darnell

Jr (1997) STATs and gene regulation. Science 277: 1630–1635.

17.

Schindler

Darnell

Jr (1995) Transcriptional responses to polypeptide ligands: The JAK-STAT pathway. Annual Review of Biochemistry 64: 621–651.

18.

Levy

Darnell

Jr (2002) Stats: Transcriptional control and biological impact. Nature Reviews Molecular Cell Biology 3: 651–662.

19.

Panigrahi

Pinder

Chan

et al . (2004) The role of PTEN and its signalling pathways, including AKT, in breast cancer; An assessment of relationships with other prognostic factors and with outcome. The Journal of Pathology 204: 93–100.

20.

Carpenter

Paw

Dewhirst

et al . (2015) Akt phosphorylates and activates HSF-1 independent of heat shock, leading to Slug overexpression and epithelial-mesenchymal transition (EMT) of HER2-overexpressing breast cancer cells. Oncogene 34: 546–557.

21.

Hafeez

Siddiqui

Asim

et al . (2008) A dietary anthocyanidin delphinidin induces apoptosis of human prostate cancer PC3 cells in vitro and in vivo: Involvement of nuclear factor-kappaB signaling. Cancer Research 68: 8564–8572.

22.

Hafeez

Mustafa

Fischer

et al . (2014) α-Mangostin: A dietary antioxidant derived from the pericarp of Garcinia mangostana L. inhibits pancreatic tumor growth in xenograft mouse model. Antioxidants & Redox Signaling 21: 682–699.

23.

Hafeez

Fischer

Singh

et al . (2015) Plumbagin inhibits prostate carcinogenesis in intact and castrated PTEN knockout mice via targeting PKCε, Stat3, and epithelial-to-mesenchymal transition markers. Cancer Prevention Research 8: 375–386.

24.

Lall

Adhami

Mukhtar

(2016) Dietary flavonoid fisetin for cancer prevention and treatment. Molecular Nutrition & Food Research 60: 1396–1405.

25.

Chen

Bao

Guo

et al . (2012) Biological activities and potential molecular targets of cucurbitacins: A focus on cancer. Anti-Cancer Drugs 23: 777–787.

26.

Jayaprakasam

Seeram

Nair

(2003) Anticancer and antiinflammatory activities of cucurbitacins from Cucurbita andreana. Cancer Letters 189: 11–16.

27.

Bartalis

Halaweish

(2011) In vitro and QSAR studies of cucurbitacins on HepG2 and HSC-T6 liver cell lines. Bioorganic & Medicinal Chemistry 19: 2757–2766.

28.

Duangmano

Sae-Lim

Suksamrarn

et al . (2012) Cucurbitacin B inhibits human breast cancer cell proliferation through disruption of microtubule polymerization and nucleophosmin/B23 translocation. BMC Complementary and Alternative Medicine 12: 185.

29.

Duncan

Alley

et al . (1996) Cucurbitacin E-induced disruption of the actin and vimentin cytoskeleton in prostate carcinoma cells. Biochemical Pharmacology 52: 1553–1560.

30.

Kim

Seo

Choi

et al . (2013) Trichosanthes kirilowii ethanol extract and cucurbitacin D inhibit cell growth and induce apoptosis through inhibition of STAT3 activity in breast cancer cells. Evidence-Based Complementary and Alternative Medicine 2013: 975350.

31.

Nakanishi

Song

et al . (2016) Autophagy is associated with cucurbitacin D-induced apoptosis in human T cell leukemia cells. Medical Oncology 33: 30.

32.

Ramani

Jacob

(2013) Prediction of P53 mutants (multiple sites) transcriptional activity based on structural (2D&3D) properties. PLoS ONE 8: e55401.

33.

Bowman

Garcia

Turkson

et al . (2000) STATs in oncogenesis. Oncogene 19: 2474–2488.

34.

Buettner

Mora

Jove

(2002) Activated STAT signaling in human tumors provides novel molecular targets for therapeutic intervention. Clinical Cancer Research 8: 945–954.

35.

Wang

Zhao

et al . (2014) Cucurbitacin IIb exhibits anti-inflammatory activity through modulating multiple cellular behaviors of mouse lymphocytes. PLoS ONE 9: e89751.

36.

Chou

(2006) Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacological Reviews 58: 621–681.

37.

Bhatia

McDonald

Jackman

et al . (1996) A simplified procedure for the purification of trichosanthin (a type 1 ribosome inactivating protein) from Trichosanthes kirilowii root tubers. Protein Expression and Purification 7: 143–146.

38.

Takahashi

Yoshida

Sugiura

et al . (2009) Cucurbitacin D isolated from Trichosanthes kirilowii induces apoptosis in human hepatocellular carcinoma cells in vitro. International Immunopharmacology 9: 508–513.

39.

Sikander

Hafeez

Malik

et al . (2016) Cucurbitacin D exhibits potent anti-cancer activity in cervical cancer. Scientific Reports 6: 36594.

40.

Ishii

Kira

Yoshida

et al . (2013) Cucurbitacin D induces growth inhibition, cell cycle arrest, and apoptosis in human endometrial and ovarian cancer cells. Tumor Biology 34: 285–291.

41.

Jacquot

Rousseau

Carbonnelle

et al . (2014) Cucurbitacin-D-induced CDK1 mRNA up-regulation causes proliferation arrest of a non-small cell lung carcinoma cell line (NSCLC-N6). Anticancer Research 34: 4797–4806.

42.

Escandell

Kaler

Recio

et al . (2008) Activated kRas protects colon cancer cells from cucurbitacin-induced apoptosis: The role of p53 and p21. Biochemical Pharmacology 76: 198–207.

43.

(2012) Cucurbitacin B regulates immature myeloid cell differentiation and enhances antitumor immunity in patients with lung cancer. Cancer Biotherapy and Radiopharmaceuticals 27: 495–503.

44.

Chen

Chiu

Nie

et al . (2005) Cucurbitacins and cucurbitane glycosides: Structures and biological activities. Natural Product Reports 22: 386–399.

45.

Kim

Lee

et al . (2010) Jnk signaling pathway-mediated regulation of Stat3 activation is linked to the development of doxorubicin resistance in cancer cell lines. Biochemical Pharmacology 79: 373–380.

46.

Barre

Vigneron

Perkins

et al . (2007) The STAT3 oncogene as a predictive marker of drug resistance. Trends in Molecular Medicine 13: 4–11.