Experimental Study Of The Inhibitory Effect Of Cucurbitacin B On Laryngeal Squamous Cell Carcinoma

IntroductionLaryngeal carcinoma ranks the 11^thcommonest kind of cancer in men worldwide, with a tendency towards an increasingly occurrence of new cases and deaths annually. Chemotherapy has been the standard approach to the treatment of patients with recurrent and metastatic squamous cell carcinoma of the head and neck and has also been confirmed as a major component of treatment for patients with locoregionally advanced,non-metastatic disease.Compared with traditional treatment with surgery followed by radiotherapy,chemotherapy given to patients with advanced laryngeal cancer allows greater organ preservation and improves survival.Cisplatin has for many years been the golden standard of chemotherapeutic agents,alone or in combination with other agents.In this regard,novel effective chemotherapeutic agents are desperately needed in the treatment of laryngeal cancer to improve survival and to enhance larynx preservation.The signal transducer and activator of transcription(STAT)proteins are so named due to the role they play in relaying signals received by specific cell surface receptors to induce transcriptional changes within the cell.So far,seven distinct STAT molecules have been identified.These include STAT 1,2,3,4,5a,5b and 6.STATs were originally discovered in the context of cytokine signalling in 1992.STAT3 plays important roles at all levels of tumorigenesis and has been identified as an oncogene. Constitutively activated STAT3 has been demonstrated in many human carcinomas. Recent in vitro and in vivo studies have demonstrated that several strategies to target STAT3 signalling have been proposed as cancer therapies and tested in preclinical studies.In addition,chemical compound screens have identified several molecules, such as cucurbitacins,that appear to specifically alter the STAT3 pathway.Cucurbitacins are compounds isolated from various plant families which have been used as folk medicines for centuries in countries such as India and China because of their wide spectrum of pharmacological activities such as cytotoxic, anti-inflammatory,and anticancer effects.Of these compounds,cucurbitacin B is one of the most widely used for in vivo and in vitro studies on tumor inhibition.Accumulated evidences have shown that cucurbitacins such as B.D.E.I.Q can inhibit the growth of numerous human cancer cell lines and tumor xenografts.Cucurbitacin B suppresses the activation of STAT3,regulates STAT3 downstream genes such as cell cycle regulating genes and apoptosis-related genes,consequently inhibits tumor growth and induces cell apoptosis.To date,however,no study has been conducted in the evaluation of the effect of cucurbitacin B on human laryngeal cancer cells.In the present study, immunohistochemistry method was used to detect the distribution and expression of p-STAT3 in 56 cases of laryngeal squamous cell carcinoma(LSCC)and 30 cases of laryngeal normal tissues.The anti-tumor effect and mechanizm of cucurbitacin B alone or incombination with docetaxcel(taxotere,TXT)on human laryngeal cancer cells were investigated in vitro and in vivo,including the effects on cell growth,cell-cycle distribution,apoptosis,and the expression of proteins relevant to the regulation of cell cycle and apoptosis pathway.Materials and methods1.The 56 cases of LSCC tissue were collected from the department of otorhinolaryngology at the Affiliated Hospital of Hangzhou Normal University. Immunohistochemistry method was used to detect expression of p-STAT3.2.Hep-2 cells were treated with different concentrations of cucurbitacin B for different time.The cell morphologic changes,cell proliferation,cell cycle distribution, and cell apoptosis were evaluated using inverted phase contrast microscope,3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay,flow cytometry,and fluorescent microscopy.The expression of p-STAT3,Bcl-2,cyclin B1 and p-ERK1/2 was evaluated by Western blot analysis.Moreover,in vivo studies were performed in a mouse xenograft model.3.Hep-2 cells were treated with cucurbitacin B alone or in combination with docetaxel.Cell growth,cell cycle distribution,and apoptosis were evaluated using MTT assay,flow cytometry,and fluorescent microscopy.The expression of p-STAT3, Bcl-2,and cyclin B1 was evaluated by Western blot analysis.In vivo inhibitory effects of cucurbitacin B in combination with docetaxel on tumor growth was evaluated in a nude mouse xenograft model.Results1.The p-STAT3 expressed both in tissues of LSCC and Laryngeal normal tissues. The expression of p-STAT3 in LSCC was higher than that in laryngeal normal tissues (P＜0.01).2.The expression of p-STAT3 was lower in 1 and 2 stages,well-differentiated and non- lymphonode metastatic LSCC than that in 3 and 4 stages(P＜0.01), poorly-differentiated LSCC(P＜0.01)and ymphonode metastatic LSCC(P＜0.05).It was not associated with the gender,age and primary location.3.Cucurbitacin B inhibited cellular proliferation in a dose and time dependent manner.One way ANOVA followed by LSD-t test showed that(P＜0.01)or P＜0.05.4.FCM analysis showed that cucurbitacin B caused an enrichment of cells in G₂/M phase in a dose and time dependent manner(P＜0.05,or 0.01 vs.control), accompanied by a reduction in G₀/G₁ phase cells(P＜0.05,or 0.01 vs.control).5.Treating Hep-2 cells with 0.1,1,and 10μM cucurbitacin B for 24 h,marked morphological changes of apoptosis such as condensation of chromatin,nuclear fragmentations and apoptotic bodies were found clearly using Hoechst 33258 staining.6.FCM analysis showed that cucurbitacin B increased the fraction of sub-G₀/G₁ peak in a dose and time dependent manner(P＜0.05,or 0.01 vs.control).7.Westen blot analysis showed that cucurbitacin B suppressed STAT3 activation in a dose and time dependent manner,subsequently decreased the level of cyclin B1 and Bcl-2 proteins and increased the level of p-ERK1/2.8.At the end of in vivo experiment,the average tumor volume of the treated groups were 0.31 + 0.03 cm³(P＜0.05),0.24 + 0.02 cm³(P＜0.01),and 0.15 + 0.01 cm³ (P＜0.01)for 27.5,55,and 110μg/kg cucurbitacin B-treated mice,respectively, significantly smaller than that of the negative control animals(0.43 + 0.05 cm³).The difference in tumor growth in these three treatment groups was significant(P＜0.05 or 0.01),indicating that cucurbitacin B inhibited Hep-2 tumor growth in vivo in a dose dependent fashion.9.Our results showed that,in comparison with single agent treatment,the combination of cucurbitacin B and docetaxel produced greater efficacy in growth inhibition,cell cycle arrest at G₂/M phase,and apoptosis induction(P＜0.05 or P＜0.01). Measuring the modulation of regulators in the cell cycle,apoptosis and signal transductions by Western blot analysis showed that the combination effect of cucurbitacin B and docetaxel was due to suppress the expression of p-STAT3,Bcl-2, and cyclin B1.10.At the end of in vivo experiment,the average tumor volume of the treated groups were 0.22+0.03 cm³(P＜0.01),0.19⁰.02 cm³(P＜0.01),and 0.11+0.01 cm³ (P＜0.01)for 55μg/kg cucurbitacin B,7.5 mg/kg doctaxel and combination-treated mice,respectively,significantly smaller than that of the negative control animals(0.44±0.05 cm³).The difference in tumor growth in combination treatment group was inhibited significantly than that in single treatment groups(P＜0.01).Conclusion1.The p-STAT3 expressed both in tissues of LSCC and Laryngeal normal tissues, however,the expression of p-STAT3 in LSCC was higher than that in laryngeal normal tissues. 2.The expression of p-STAT3 in LSCC was associated with the clinic stage, differentiation degree and metastatic.It was not associated with the gender,age and primary location.3.Cucurbitacin B inhibits the growth of LSCC in vitro and in vivo.4.The antitumor effect of cucurbitacin B on Hep-2 cells was due to the induction of cell cycle arrest as well as apoptosis.5.The possible mechanisms underlying the action might be attributed to the suppression of STAT3 activation.Subsequently decreased the level of cyclin B1 and Bcl-2 proteins and increased the level ofp-ERK1/2.6.Cucurbitacin B in combination with docetaxel had synergistic antitumor activity against LSCC in vitro and in vivo.7.The anti-tumor efficacy mediated via the combination of cucurbitacin B and docetaxel was mainly due to the induction of cell cycle arrest as well as apoptosis.8.The possible mechanisms underlying the antitumor activity of cucurbitacin B in combination with docetaxel might be attributed to the suppression of STAT3 activation, subsequently decreasing the expression level of cyclin B1 and Bcl-2 proteins.The detail mechanism by which cucurbitacin B enhances the antitumor effects of docetaxel on LSCC needs futher investigation.