| BackgroundHypopharyngeal squamous cell carcinoma(HSCC)is one of the most aggressive malignancies in head and neck squamous cell carcinoma(HNSCC).HSCC has the tendency to spread submucosally and patients with HSCC usually present asymptomatically until they are diagnosed in the advanced stage.Additionally,HSCC is characterized by poor histological differentiation and frequent lymph node metastasis.Thus,prognoses of HSCC patients are quite poor,and the 5-year survival rate is approximately 20-40%.Currently,the majority of HSCC patients are treated with surgery followed by postoperative radiotherapy or concurrent chemoradiation.Cisplatin-based chemotherapy regimen is partly beneficial for preserving laryngeal function and prolonging survival of patients,but HSCC itself still remains insensitive to cisplatin and is inclined to develop resistance during chemotherapy.Therefore,it is of vital significance to search for new agents or approaches in order to improve the sensitivity of HSCC to chemotherapy.Myeloid cell leukemia-1(Mcl-1)is an important anti-apoptotic member of B-cell lymphoma-2(Bcl-2)family.Mcl-1 is found widely upregulated in HNSCC,and its high expression is associated with tumor cell survival and resistance to chemo-or radiotherapy of HNSCC cells.Increasing evidence shows that induction of apoptosis in HNSCC by drugs or other factors was closely linked to downregulation or dysfunction of Mcl-1.In addition,decrease of Mcl-1 using antisense RNA technology sensitized HNSCC cells to cisplatin or other chemotherapeutic drugs.These findings suggest that we could involve agents targeting Mcl-1 in the treatment of HSCC.In recent years,one innovate way to downregulation of Mcl-1 through inhibition of its transcription has been proposed by some researchers.RNA polymerase II(RNAP Ⅱ)plays a vital role in initiation and elongation processes during genes’transcription,such as Mcl-1.Activation of RNAP II is highly regulated by a sequence of events,among which the positive transcriptional elongation factor b(p-TEFb)exerts critical effects by phosphorylating the serine-2 of the carboxyterminal domain heptapeptide repeats of RNAP Ⅱ(p-RNAP ⅡS2).Cyclin-dependent kinase 9/cyclin T(CDK9/cyclin T),a key component of RNAP Ⅱ,is responsible for the phosphorylation,making itself a target for inhibiting Mcl-1 transcription.Over the last decade,several CDK9 inhibitors have been developed,including CDKI-73.CDKI-73 synthesized by Prof.Shudong Wang from South Australia University is one of the most potent CDK9 inhibitors identified to date(Ki =3 nM).Additionally.CDKI-73 has several advantages over other CDK9 inhibitors,i.e.low cytotoxity to normal cells and more specific inhibition of CDK9.In assessment of the efficacy of CDKI-73 in chronic lymphoid leukemia,acute myeloid leukemia and ovarian cancer,CDKI-73 was highly cytotoxic to tumor cells,but showed a low toxicity against normal cells.Currently.Prof.Shudong Wang is preparing for its clinical trial application in both Australia and China.Our previous data showed that Mcl-1 was actively transcribed in HSCC and its mRNA expression was positively associated with tumor size,which is in consistency with HNSCC,suggesting that it might play an important role in maintaining cell survival of HSCC and decreasing its sensitivity to cisplatin.On the basis of that,we hypothesized that CDK9 inhibitors may be effective in inhibiting proliferation and increasing chemosensitivity of HSCC.However,application of CDK9 inhibitors in HSCC have not yet been reported.Thus,in this study,we evaluated the effect of CDKI-73 on cell survival and chemosensitivity of HSCC through in vitro experiments,and preliminarily investigated its underlying mechanism.Objectives1.To identify the potential role of CDKI-73 in treatment of HSCC through detection of Mcl-1 transcription status in HSCC tumor tissues versus(vs.)adjacent normal mucosae.2.To explore the effect of CDKI-73 on proliferation of HSCC cells and the relevant mechanism.3.To investigate whether CDKI-73 can sensitize HSCC cells to cisplatin treatment.Methods and results1.Compared with adjacent normal mucosae,the mRNA level of Mcl-1 is upregulated in HSCC tumor tissuesTo observe the transcription status of Mcl-1 in HSCC tumor tissues,we extracted total RNA from HSCC tumor tissues(n = 57)and adjacent normal mucosae(n = 31).Real-time PCR assay revealed that Mcl-1 mRNA levels were significantly higher in tumor tissues compared to those in adjacent non-tumor mucosae(p<0.01),regardless of tumor locations,indicating that Mcl-1 was actively transcribed in HSCC tumor tissues.Further,we analyzed the relationship between Mcl-1 mRNA levels with clinicopathological characteristics of patients,finding that the levels of Mcl-1 mRNA in tuimor tissues positively corresponded with tumor sizes(p= 0.01)and clinical stages of the patients(p = 0.017).2.CDKI-73 inhibits proliferation of FaDu cells in vitroWe next evaluated the pharmacological effect of CDKI-73 on FaDu cells i.e.human HSCC cells.After FaDu cells were treated with CDKI-73 for 24 or 48 h,cell viability was measured though CCK8 assays.The result showed that cell viability of FaDu cells decreased in a time or dose dependent manner.However,in the same condition,HEK293 cells,normal human embryonic kidney cells were significantly less susceptible to CDKI-73(p<0.05 orp<0.01).Additionally,IC50s of FaDu vs.HEK293 cells after exposure to CDKI-73 for 24 and 48 h,were 5.77 ± 2.87μM vs.66.96 ± 25.66 μM(p = 0.01)and 0.86 ± 0.33 μM vs.8.82 ± 4.64 μM(p<0.01),respectively,suggesting that CDKI-73 preferentially targeted HSCC cells over non-transformed cells.3.CDKI-73 exerts anti-proliferative effects through inducing apoptosis and causing cell cycle arrest in FaDu cellsThen,we explored whether CDKI-73 exerted anti-proliferative effects through inducing apoptosis in FaDu cells.Caspase-3 activity detection assays showed that caspase-3 in FaDu cells was activated in a dose dependent manner after exposure to CDKI-73 for 48 h.As the target of caspase-3,cleavage of poly(ADP-ribose)polymerase(PARP)was also enhanced with the increase of dosage of CDKI-73.Additionally,the percentages of apoptotic FaDu cells stained by Annexin V-FITC/PI increased time-and dose-dependently and nearly reached the inhibitory rates in CCK8 assays under the same treatment.These data indicated that CDKI-73 inhibited growth of FaDu cells mainly through inducing apoptosis.The pro-apoptotic role of CDKI-73 was further verified in Detroit 562 cells i.e.one human pharynx carcinoma cell line,which was derived from pleural effusion.Then,we studied whether the anti-proliferative property of CDKI-73 in FaDu cells was associated with cell cycle effect.After being stained by PI?cell cycle distribution of FaDu cells was detected by flow cytometry,demonstrating that low dosage(i.e.0.25 μM)of CDKI-73 caused cell cycle arrest at the G0/G1 phase in comparison with the untreated control(65.18 ± 3.12%vs.46.13 ± 2.22%,p<0.01).Thus,we thought cell cycle arrest caused by CDKI-73 might also contribute to its inhibitory effect on growth of FaDu cells.4.The cellular mode of action(MoA)of CDKI-73 in FaDu cellsFurthermore,we explored the mode of action of CDKI-73 in the cellular level.After FaDu cells were treated with a series of dosages of CDKI-73 for 24 h,Western blot assays showed that phosphorylation of CDK9 was obviously inhibited and asbiomarker of CDK9 activity,p-RNAPⅡS2 was also markedly reduced.Additionally,expression of Mcl-1 was decreased dose-dependently and even abrogated.Further,transcription of Mcl-1,Bcl-2 and X-linked inhibitor of apoptosis protein(XIAP)in FaDu cells demonstrated by Real-time PCR assays were inhibited dose-dependently after exposure to CDKI-73 for 12 h.The transcriptional inhibition of Mcl-1 was most remarkable,followed by Bcl-2.Likewise,the inhibitory effect of CDKI-73 on transcription was confirmed in Detroit 562 cells and apparently,Mcl-l was a preferential target of CDKI-73.To ascertain that Mcl-1 was more susceptible to CDKI-73,we measured the mRNA levels of Mcl-1 and Bcl-2 in FaDu cells at different time points after exposure to CDKI-73 at a low concentration(i.e.0.5μM),using Real-time PCR assays.A time-dependent decrease of Mcl-1 and Bcl-2 mRNA levels were significantly observed and CDKI-73 again preferentially targeted McI-1 over Bcl-2 at each time point(p<0.01).These data collectively demonstrated that CDKI-73 could downregulate transcription and expression of Mcl-1 in FaDu cell,and Mcl-1 was its preferential target.5.The MoA of CDKI-73 in FaDu cells may contribute to CDKI-73 mediated apoptosisTo ascertain that CDK9 is the target via which CDKI-73 induces apoptosis in FaDu cells,we transfected FaDu cells with siRNAs(i.e.S1 and S2)targeting CDK9 or scrambled nucleotide(SN).As demonstrated by Western blot and Real-time PCR assays,silencing of CDK9 inhibited p-RNAP Ⅱs2 and subsequent transcription and expression of Mcl-1,implying indirectly that CDKI-73 functioned by targeting CDK9.More importantly,flow cytometry showed that depletion of CDK9 significantly caused apoptosis in FaDu cells in comparison with negative control after being stained by Annexin V-FITC/PI(S1vs.SN,19.69 ± 4.09%vs.9.49 ± 2.53%,p<0.05;S2vs.SN,22.25 ± 3.4I%vs.9.49 ± 2.53%.p<0.05),suggesting that CDKI-73 might promote apoptosis through targeting CDK9.It has been shown that FaDu cells are dependent on Mcl-1 for survival.Thus,we think downregulation of Mcl-1 through inhibiting CDK9 was probably the mechanism underlying that CDKI-73 promotes apoptosis in FaDu cells.6.CDKI-73 sensitized FaDu cells to cisplatinUpregulation of Mcl-1 in HNSCC contributes greatly to chemoresistance of HNSCC.Considering that CDKI-73 has a role in downregulating transcription and expression of Mcl-1,we further explored whether CDKI-73 could improve sensitivity of HSCC cells to cisplatin.In CCK8 assays,FaDu cells were treated with a series of cisplatin alone or in combination with a moderate concentration of CDKI-73(i.e.0.25 UM),which could suppress Mcl-1 expression but not induce too much apoptosis.The result showed that the inhibitory effect caused by each dosage of cisplatin alone was significantly weaker than combination of the corresponding dosage of cisplatin and 0.25μM of CDKI-73(p<0.01).By means of CompuSyn program,we obtained combination indexes(CIs)of the above different combination regimens.Their CIs were all less than 1,meaning that the two agents might act synergistically against proliferation of FaDu cells.In other words,CDKI-73 could increase the sensitivity of FaDu cells to cisplatin.Additionally,the synergism was verified by Annexin V/PI dual staining of FaDu cells after being treated with the combination of CDKI-73(0.25 μM)and cisplatin(1μM),leading to 54.4 ± 8.3%apoptotic cells in comparison with exposure to either CDKI-73(20.6 ± 3.0%,p<0.01)or cisplatin alone(24.7 ± 5.9%,p<0.01).Conclusions1.Mcl-1 is actively transcribed in HSCC,and its mRNA levels are closely correlated with tumor sizes and clinical stages of HSCC patients,suggesting that Mcl-1 may be implicated in survival maintenance or chemoresistance of HSCC cells and CDK9 inhibitors may be valuable in HSCC treatment.2.CDKI-73 can preferentially inhibit proliferation of FaDu cells over non-transformed cells mainly through induction of apoptosis.Additionally,cell cycle arrest caused by CDKI-73 may partly contribute to its anti-proliferative effect.These findings indicate that CDKI-73 alone might be effective in treating HSCC.3.Downregulation of Mcl-1 transcription and expression might be the mechanism through which CDKI-73 induces apoptosis in FaDu cells.4.In vitro experiments confirmed that CDKI-73 synergized with cisplatin against FaDu cells,suggesting that CDKI-73 can potentiate the effect of cisplatin against FaDu cells. |
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