Apoptosis of colon cancer CT-26 cells induced polysaccharide from Cyclocarya paliurus and its phosphorylated derivative via intrinsic mitochondrial passway

In this study,the antitumor properties and the possible molecular mechanisms of Cyclocarya paliurus polysaccharide(CP)and its phosphorylated derivative(P-CP)on CT-26 mouse colon carcinoma cells were investigated.Results found that CP had high inhibition ratio against CT-26 cells.The flow cytometry results found that CP treatment could cause the intracellular acidification,arrest the cell cycle in the S phase and increase reactive oxygen species generation.Additionally,CP treatment triggered mitochondrial membrane potential depolarization and Ca^(2+)overloading,and broke down the balance of antioxidant system,Na^(+)/K^(+)-ATPase and Ca^(2+)-ATPase.Further analysis found CP induced cell apoptosis through improving the activities of caspase-3 and caspase-9,and increasing the level of cytochrome C.Furthermore,the comparative study of antitumor effect on CT-26 cells displayed that the phosphorylation enhanced antitumor activities of polysaccharides.These results suggest CP is a potential natural therapeutic agent for colon cancer and phosphorylation represents an effective method of enhancing the antitumor activity of CP.

Environmental hypoxia induces apoptosis in large yellow croaker Larimichthys crocea via both intrinsic and extrinsic pathways

Hypoxia has become an unfavorable factor affecting the sustainable development of the large yellow croaker Larimichthys crocea,an economically important mariculture fish in China.Apoptosis is a consequence of hypoxia on fish.However,the effects of hypoxia stress on apoptosis in L.crocea remain largely unknown.We investigated the effect of environmental hypoxia on apoptosis in L.crocea.Results show that hypoxia induced apoptosis in L.crocea both in vivo and in vitro.The mitochondrial membrane potential was significantly reduced in large yellow croaker fry(LYCF)cells.The expression levels of Bcell lymphoma/leukemia-2(Bcl-2)m RNA and protein were also significantly decreased in the liver and LYCF cells during 96 h and 48 h of hypoxia stress,respectively,whereas the expression level of Bcl-2 associated X(Bax)mRNA,Casp3 mRNA,and activity of caspase-3/7/9 were significantly increased,indicating that hypoxia induced caspase-dependent intrinsic apoptosis in L.crocea.The expression level of the apoptosis-inducing factor(AIF)protein was significantly increased in the liver and LYCF cells.The level of AIF protein was significantly decreased in the cytoplasm but increased in the nuclei of L.crocea,demonstrating that hypoxia induced the AIF-mediated caspase-independent intrinsic apoptosis.In addition,the activity of caspase-8 was significantly increased,indicating that hypoxia stress induced extrinsic apoptosis in L.crocea.Therefore,hypoxia induced apoptosis in L.crocea through both the intrinsic and extrinsic pathways.The present study accumulated basic biological information to help elucidate the mechanism of hypoxia response in marine fish.

Essential Oil from Siegesbeckia pubescens Induces Apoptosis through the Mitochondrial Pathway in Human HepG2 Cells

Siegesbeckia pubescens（SP） has been used as a traditional medicine for the treatment of and inflammatory diseases. However, the activities of SP against hepatocellular carcinoma and the related mechanisms remain unclear. The present study aimed to examine the effects of the essential oil of SP（SPEO） on the proliferation of hepatocellular carcinoma cells and the possible mechanisms. The growth inhibition of Hep G2 cells was analyzed by MTT assay. Hoechst 33258 and fluorescence microscopy were utilized to observe the nuclear morphological changes of apoptotic cells. Flow cytometry was used to detect cell apoptosis and cell cycle. The expressions of the target proteins were detected by Western blotting. The results showed that SPEO obviously inhibited the proliferation of Hep G2 cells in a dose-dependent manner. SPEO activated a series of apoptotic proteins in Hep G2 cells, increasing expression levels of Bax, caspase-3 and caspase-9, and decreasing the bcl-2 expression level. SPEO displayed promising anti-hepatocellular carcinoma activities in vitro, partly by inducing apoptosis in Hep G2 cells through activating the mitochondrial pathway.

Yi Qi Jie Du Decoction Inhibits Proliferation and Induces Apoptosis of Nasopharyngeal Carcinoma Stem Cells Through Mitochondrial Apoptosis Pathway

Objective To assess the effects of Yi Qi Jie Du Decoction(YQJDD)on nasopharyngeal carcinoma stem cells(NPC-SCs)and investigate the underlying mechanism.Methods NPC-SCs were collected in serum-free culture system and identified by the sphere formation assay.The effect of YQJDD on the proliferation of NPC-SCs was detected using the Cell Counting Kit-8 assay.The change of mitochondrial membrane potential(ΔΨm)in NPC-SCs following treatment with YQJDD was investigated by JC-1 staining.The levels of apoptosisassociated proteins were examined using western blot analysis.Results YQJDD significantly inhibited the proliferation of NPCSCs in dose-and time-dependent manners.JC-1 staining revealed that YQJDD lowered theΔΨm of NPC-SCs in a timedependent manner.After treatment with YQJDD,the expression of cleaved caspase-3,-7 and-9,cleaved poly-ADP ribose polymerase,P21 and P53 were increased,while the expression of Survivin in NPC-SCs was decreased.However,the expression of cleaved caspase-8 remained nearly unchanged.Conclusions YQJDD inhibits the growth and induces apoptosis of NPC-SCs via an intrinsic apoptosis pathway.

The study on DNA methylation of p53-Bax mitochondrial apoptosis pathway in cholangiocarcinoma

Objective： To study the methylation status of several genes on p53-Bax mitochondrial apoptosis pathway and clinical significance in cholangiocarcinoma. Methods： Promoter hypermethylation of DAPK, p14 and ASC genes were detected by methylation-specific PCR. p53 gene status （exon 5-8） were examined by automated sequencing, combined with the clinical documents of patients by statistics analysis. Results： （1） We found 66.7% of 36 cases cholangiocarcinoma had methylation of at least one tumor suppressor gene. The frequency of tumor suppressor gene methylation in cholangiocarcinoma was： p14 （24%）, DAPK （30.6%）, TMSI/ASC （36.1%）. The frequency of tumor suppressor gene methylation in tissues near cancer was： DAPK （5.6%）, TMS1/ASC （8.3%）. （2） p53 gene mutations were found in 22 of 36 patients （61.1%）. （3） There were no statistically relationship among the methylation of DAPK, p14 and ASC genes. There were negative relationship differences between the methylation of p14 and p53 gene mutation （P 〈 0.05）. （4） p53 gene mutation combined with the methylation of tumor suppressor were 14 cases （38.9%）. There were statistically differences on extent of pathologic biology, differentiation and invasion （P 〈 0.05）. Conclusion： Our study indicated that methylation of p53-Bax mitochondrial apoptosis pathway in cholangiocarcinoma was a common epigenetic event. Although the methylation of ASC, DAPK genes was low, it might be significance for early diagnosis, p53 gene mutation combined with the methylation of tumor suppressor might be relationship with pathologic biology, it trended to more malignancy.

HSP70 inhibitor combined with cisplatin suppresses the cervical cancer proliferation in vitro and transplanted tumor growth:An experimental study

Objective:To study the regulating effect of HSP70 inhibitor(PES) combined with cisplatin on cervical cancer proliferation in vitro and transplanted tumor growth.Methods:Cervical cancer Hela cell lines were cultured and divided into control group,cisplatin group,PES group and cisplatin+PES group that were treated with serum-free DMEM,cisplatin with final concentration of 10 μmol/L,PES 20 μmol/L and cisplatin 10 μmol/L combined with PES with 20 μmol/L,respectively;animal models with cervical cancer xenografts were established and divided into control group,cisplatin group,PES group and cisplatin+PES group who received intra-tumor injection of normal saline,10 μmol/L cisplatin,20 μmol/L PES as well as 10 μmol/L cisplatin+20 μmol/L PES,respectively.Cell proliferation activity,transplanted tumor volume and mitochondria apoptosis molecule expression were detected.Results:Cell viability value and Bcl-2 mRNA expression in cells of cisplatin group,PES group and cisplatin+PES group were significantly lower than those of control group while Bax,Caspase-3 and Caspase-9 mRNA expression in cells were significantly higher than those of control group;transplanted tumor volume and the Bcl-2 mRNA expression in transplanted tumor tissue of cisplatin group,PES group and cisplatin+PES group were significantly lower than those of control group while Bax,Caspase-3 and Caspase-9 m RNA expression in transplanted tumor tissue were significantly higher than those of control group.Conclusions:HSP70 inhibitor combined with cislatin can inhibit cervical cancer cell proliferation in vitro and transplanted tumor growth through mitochondrial apoptosis pathway.

Combination of Total Astragalus Extract and Total Panax Notoginseng Saponins Strengthened the Protective Effects on Brain Damage through Improving Energy Metabolism and Inhibiting Apoptosis after Cerebral Ischemia-Reperfusion in Mice

Objective： To explore the effects and molecular mechanisms of the combination between total Astragalus extract （TAE） and total Panax notoginseng saponins （TPNS） against cerebral ischemia- reperfusion injury. Methods： C57BL/6 mice were randomly divided into sham-operated group, model group, TAE （110 mg/kg） group, TPNS （115 mg/kg） group, TAE-TPNS combination group and Edaravone （4 mg/kg） group, treated for 4 days, then, cerebral ischemia-repeffusion injury was established by bilateral common carotid artery （CCA） ligation for 20 min followed by reperfusion for 1 and 24 h. Results： TPNS could increase adenosine triphosphate （ATP） level, TAE and TAE-TPNS combination increased ATP, adenosine diphosphate （ADP） contents and Na＋-K＋-ATPase activity, and the effects of TAE-TPNS combination were stronger than those of TAE or TPNS alone after reperfusion for 1 h. After reperfusion for 24 h, TAE, TPNS and TAE-TPNS combination significantly increased neurocyte survival rate and decreased the apoptosis rate as well as down-regulated the expression of phosphorylated c-June N-terminal kinasel/2 （p-JNK1/2）, cytochrome C （Cyt C）, cysteine aspartic acid-specific protease （Caspase）-9 and Caspase-3. Furthermore, the effects in TAE-TPNS combination were better than those in TAE or TPNS alone. Conclusion： The combination of TAE 110 mg/kg and TPNS 115 mg/kg could strengthen protective effects on cerebral ischemia injury, the mechanism underlying might be related to improving jointly the early energy metabolism, and relieving the delayed apoptosis via inhibiting the mitochondrial apoptosis pathway of JNK signal transduction.

Xuezhikang(血脂康) Reduced Renal Cell Apoptosis in Streptozocin-Induced Diabetic Rats through Regulation of Bcl-2 Family

Objective： To investigate the effect of Xuezhikang（血脂康, XZK） on renal cell apoptosis in diabetic rats and the possible mechanism. Methods： Sixty-six rats were randomly divided into 3 groups： the normal, model and XZK groups. In each group, the rats were further randomly divided into 3-month and 6-month subgroups, respectively. Diabetes of rats was induced by a single intraperitoneal injection of 1% streptozocin at 60 mg/kg body weight. Rats in the XZK group received gastric perfusion of XZK（1200 mg/kg body weight） everyday for 3 or 6 months, while rats in the normal and model groups received equal volume of saline. Twenty-four hours＇ urine was collected for urinary albumin excretion（UAE） measurement. Periodic acid-Schiff（PAS） and Masson＇s trichrome staining were used for saccharides and collagen detection. Cell apoptosis of renal cortex was investigated by Td T-mediated d UTP nick end labeling（TUNEL） staining. Bax and Bcl-2 expressions were detected by immunohistochemistry and Western blot, respectively. Cytochrome C（Cyt C） and caspase-9 concentration were detected by Western blot. Results： Compared with the model group, XZK treatment could significantly decrease the kidney hypertrophy index, 24 h UAE, renal cell apoptosis, cytoplasmic Cyt C level and active caspase-9 level, as well as suppress the increment of Bax and up-regulate the expression of Bcl-2, leading to the suppression of Bax/Bcl-2 ratio at 3 and 6 months（P〈0.05 or P〈0.01）. Moreover, XZK treatment could alleviate the deposition of PAS-stained saccharides and Masson＇s trichromestained collagen to different extent. Conclusions： Renal cell apoptosis was observed in diabetic kidney, in which mitochondrial apoptotic pathway might be involved. XZK treatment could attenuate pathological changes in diabetic kidney and reduce renal cell apoptosis, probably via the suppression of Bax/Bcl-2 ratio, which lead to inhibition of Cyt C release and following caspase-9 activation.