| Introduction:Based on GLOBOCAN estimates,bladder cancer is the sixth most diagnosed cancer in men and the fifteenth most diagnosed cancer in women,with approximately 429,800 new cases and 165,100 deaths in 2012 worldwide.The mucosa or lamina propria(Ta and T1)account for about 70%of newly diagnosed cases.The current gold standard for Ta and T1 bladder preservation treatment is transurethral resection of bladder tumour(TURBT).However,TURBT alone is not adequate therapy for superficial bladder cancer.50%to 70%of Ta/T1 bladder cancer cases recur within 5 years after TURBT.In addition,5%to 20%of cases develop into pathologically progressive disease.Therefore,postoperative adjuvant therapy is necessary and the most effective agent is Bacillus Calmette-Guerin(BCG).However,BCG is ineffective in 30-40%of Normuscle Invasive Bladder Cancer(NMIBC)patients,and also 35%of the initial responders relapse within 5 years.Thus,new treatment options are required for the efficient treatment of bladder cancer.Lycorine is a pyrrolo[de]phenanthridine ring-type alkaloid extracted from Amaryllidaceae genera,whose structure was firstly determined by Nagakawa et al.In 1956,Lycorine possesses various biological effects including a ntiviral,antimalarial and antiinflammation.Furthermore,it has been proved that lycorine has the inhibition of the growth of a variety of tumors,such as leukemia,cervical cancer and prostaticcancer.However,there are still no reports on the effect of lycorine on bladder cancer.The PI3K family participates in multiple signaling pathways to regulate cellular functions.Moreover,PI3K/Akt signaling pathway plays an important role in tumorigenesis and development.The lipid products,PI(3,4)P2 and PI(3,4,5)P3,produced by PI3K act as the second messengers by binding and activating the intracellular target prote ins to form a signal transduction cascade,and finally adjust proliferation,differentiation,survival,and migration of cells.Akt,also known as PKB or Rac,plays a critical role in regulation of survival and apoptosis.The activity o f this protein kinase is regulated by insulin and various growth and survival factors to function in a wortmannin-se nsitive pathway involving PI3 kinase.The activation of Akt results from both phosphorylation within binding and ac tivation loop at Thr308 by PDK1 and phosphorylation within the carboxy terminus at Ser473.phosphorylation of b oth residues is critical to generate a high level of akt activity,and the phosphorylation of Thr3 08 is not dependent on phosphorylation of Ser473 or vice versa.Akt promotes cell survival by inhibiting apoptosis through phosphoryl ation and inactivation of several targets,including Bax,forkhead transcription factors,MDM2,and caspase-9.PTEN phosphatase is a major negative regulator of the PI3K/Akt signaling pathway.This study was aimed to illustrate the cytotoxic effects of Lycorine on human bladder cell,T24 cells.We have further explored the changes of apoptosis related proteins such as pten,akt,bax,bcl-2,and the critical factor caspase-3.Herein,our study demonstrated that Lycorine showed cytotoxicity,decreased the expression of critical factor p-akt by increasing the expression of its negative regulator pten,and finally increased the expression of the critical executioner of apoptosis cleaved-caspase-3.In addition,the fact that Lycorine show little cytotoxic effect on normal cells Svhuc suggests that Lycorine may be a safe and efficient medical treatment for bladder cancer the rapy.In this work,we provided a rational for the further preclinical and clinical evaluation of Lycorine for bladder cancer therapy.Results:1.Lycorine induced apoptosis of T24 cell lines.previous studies hace shown that Lycorine inhibited the growth of various malignant cells.To determine whether Lycorine inhibited the growth of T24 cells.The MTS assays showed that Lycorine significantly inhibited the growth of T24 cells,which indicated that Lycorine has antitumor activity on bladder cancer.The IC50 values of lycorine on T24 cells for 12h,24h,48h,72h were 46.9,14.7,7.5,3.6(uM),and The IC50 values of lycorine on T24 cells for 24h,48h were 39.1,23.4(uM).3.2 Apoptosis was detected using Hoechst33342 staining assay.Apoptotic nucleus shrank,showed condensed or fragmented fluorescence.there are few apoptotic cells in empty group.In 25nM T24 group,there are about 46%of apoptotic cells in whole cells.In 25nM svhuc group,there are about 5%of apoptotic cells in whole cells.Lycorine significantly increased the percentage of apoptotic cells in T24 cells,which indu ced the little apoptosis-inducing effect in normal bladder cancer-svhuc cells.2.There is evidence that Lycorine induced T24 cells apoptosis and it has been suggested that the apoptosis in many tumors results from inhibiting Akt and activating the intrinsic caspase cascade.In order to determine differences in the PI3K-Akt-signalling-pathway in T24 cells before and after using Lycorine,relevant mRNA and protein expression were determined by qPCR and WB,respectively.Expression levels were determined upon unused-Lycorine T24 cells.Akt mRNA expression has nonsignificant change in each groups,and little significant change was detected in Akt protein expression.However,phospho-Akt(Ser473)protein levels were reduced in both 25uM and 50uM group contrasted with blank group,which means that Lycorine’s apoptosis-inducing effect operated by phosphorylation.Correspondingly,the negative regulator of P-Akt,PTEN protein expression increased significantly in both 25uM and 50uM group.In terms of other signal molecules,caspase-3 and bax mRNA expression had a significant increase in 50uM group in comparison with blank group by qPCR,and bcl-2 m RNA expression was decreased in 50uM group compared with blank group.Lycorine downregulated Caspase-3 protein expression while upregulated caspase-3 mRNA expression,but a significant increase of cleaved-caspase-3 protein expression was detected in 50uM group,which is well known as the activated fragment of caspase-3.Immunofluorescence staining showed that cleaved-caspase-3 was increased significantly after treatment with Lycorine in the cytoplasm,and phospho-Akt(Ser473)was decreased after treatment with Lycorine both i n the cytoplasm and enucleus.3.T24 cells were injected into nude mice to establish the T24 subcutaneous tumor xenograft model.Mice we re divided into 3 groups(n=5 per group)and treated with Lycorine at 5 mg/kg/day or 10 mg/kg/day or vehicle control.At the day 18,mice were sacrificed;tumor xenografts were dissected and the tumor weights were calculated.It could be concluded that Lycorine significantly suppressed the tumor growth of T24.The average tumor weight was 1137.63±183mg for PBS group,415.100±37mg for 5mg/k g/day group and 293.44±47mg for 10 mg/kg/day group,respectively.And statistical result showed significant difference between the drug-treated groups and the control group.Discussion:Despite bladder cancer being the fifth most common cancer in the United States,there have been few advances in its treatment.Minimal progress has been made in the treatment of bladder cancer in over a decade.NMIBC should be treated with TURBT and intravesical adjuvant chemotherapy,but chemotherapy has its known side effects such as chronic bladder imflammaiton which impacts patient life.Our principle findings in this study show that the anti-tumor activity of Lycorine toward T24 is reliable.We present that Lycorine induces apoptosis and inhibits proliferation and in T24 cells.Lycorine downregulated the expression of p-Akt.Adminstration of Lycorine delays tumor growth and reduces both tumor weight and volume.All these results provide a characterization of the unique anti-tumor properties of Lycorine as a potential candidate for the treatment of bladder cancer.The anti-tumor effect of Lycorine has been proved in various tumors.For example,the Up-regulation of p21 and TNF-alpha is mediated in Lycorine-induced death of HL-60 cells.Lycorine induces apoptosis and down-regulation of Mcl-1 in human leukemia cells.Furthermore,Lycorine induces programmed necrosis in the multiple myeloma cell line ARH-77.However,the anti-tumor effect of Lycorine on bladder cancer cells has not yet been investigated.Thus,the present study is the first to show the suppression of P-Akt,and the activation of intrinsic caspase pathway,for the induction of bladder cancer apoptosis by Lycorine.Interestingly,we also investigated the effect of Lycorine on normal bladder cancer Svhuc cells.The number of apoptotic nucleus presents that the Svhuc cells inhibitory rate was significantly less than T24 cells.The results may be due to the characteristics of bladder cancer.Previous studies have reported that bladder cancer has a high mutation rate,exceeded only by lung cancer and melanoma for the reason that genetic alterations,such as mutations,copy number alterations or RNA expression changes in PI3K/AKT/mTOR pathway are present in more than 40%of urinary bladder cancer.Moreover,loss or reduced expression of PTEN is rare in NMIUBC,but it frequently occurs in MIUBC.It should be noted that these results were measured in tumors obtained from only one bladder cancer cell lines.To better utilize Lycorine’s therapeutic potential,further investigations are needed to be performed on other types of urinary bladder cell lines.besides,we did not interfere Akt by small interfering RNA.Though the more imperative exploration is needed,these data that we obtained have suggested that PI3K/Akt pathway were involved in the inhibition of proliferation and survival caused by Lycorine,especially in the T24 bladder cancer. |
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