Glycogen synthase kinase-3β positively regulates the proliferation of human ovarian cancer cells

Although glycogen synthase kinase-3 （GSK-3） might act as a tumor suppressor since its inhibition is expected to mimic the activation of Wnt-signaling pathway, GSK-3β may contribute to NF-κB activation in cancer cells leading to increased cancer cell proliferation and survival. Here we report that GSK-3β activity was involved in the proliferation of human ovarian cancer cell both in vitro and in vivo. Inhibition of GSK-3 activity by pharmacological inhibitors suppressed proliferation of the ovarian cancer cells. Overexpressing constitutively active form of GSK-3β induced entry into the S phase, increased cyclin D1 expression and facilitated the proliferation of ovarian cancer cells. Furthermore, GSK-3 inhibition prevented the formation of the tumor in nude mice generated by the inoculation of human ovarian cancer cells. Our findings thus suggest that GSK-3β activity is important for the proliferation of ovarian cancer cells, implicating this kinase as a potential therapeutic target in ovarian cancer.     

Antitumor effect of antisense ornithine decarboxylase adenovirus on human lung cancer cells

Ornithine decarboxylase (ODC),the first enzyme of polyamine biosynthesis,was found toincrease in cancer cells,especially lung cancer cells.Some chemotherapeutic agents aimed at decreasingODC gene expression showed inhibitory effects on cancer cells,ha this study,we examined the effects ofadenoviral transduced antisense ODC on lung cancer cells.An adenovirus carrying antisense ODC(rAd-ODC/Ex3as) was used to infect lung cancer cell line A-549.The 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay was used to analyze the effect on cell growth.Expression of ODC and concentrationof polyamines in cells were determined by Western blot analysis and high performance liquid chromatography.Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling was used to analyze cellapoptosis.The expression of ODC in A-549 cells was reduced to 54%,and that of three polyamines wasalso decreased through the rAd-ODC/Ex3as treatment.Consequently,cell growth was substantially inhibitedand terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling showed that rAd-ODC/Ex3ascould lead to cell apoptosis,with apoptosis index of 46%.This study suggests that rAd-ODC/Ex3as has anantitumor effect on the human lung cancer cells.     

Effects of basic fibroblast growth factor on angiogenin expression and cell proliferation in H7402 human hepatoma cells

Hepatocellular carcinoma （HCC） is one of the most common cancers worldwide. Basic fibroblast growth factor （bFGF）, which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Its expression is essential for the progression and metastasis of HCC. This study aims to investigate the effects of bFGF on the expression of angiogenin, another growth factor, which plays an important role in tumor angiogenesis, and on cell proliferation in H7402 human hepatoma cells. The bFGF sense cDNA or antisense cDNA was stably transfected into H7402 cells. Genomic DNA PCR analysis demonstrated that human bFGF sense cDNA or antisense cDNA was inserted into the genome. Furthermore, the expression of bFGF and angiogenin was examined by RT-PCR and Western blot assays. MTT and colony formation assays were employed to determine cell proliferation. Stable bFGF over-expressing and under-expressing transfectants were successfully established. Expression of angiogenin was decreased in the over-expressing bFGF cells （sense transfectants） and was increased in the under-expressing bFGF cells （antisense transfectants）. Cell proliferation increased in the bFGF sense transfectants and decreased in the bFGF antisense transfectants. These results demonstrated that the endogenous bFGF may not only negatively regulate the angiogenin expression but also contribute to the overall cell proliferation in H7402 human hepatoma cells. This study may be helpful in finding a potential therapeutic approach to HCC.     

Trop-2 targeted CAR-T cells inflict enhanced killing of ovarian cancer cells

T cell modified by chimeric antigen receptor (CAR) is considered one of the most promising tumor therapies for its almost magic effect in the treatment of hematologic malignancies. Encouraged by breakthroughs in this area, we produced Trop-2-redirected chimeric antigen receptor-modified T (Trop-2 CAR-T) cells and tested their function on ovarian cancer cells in vitro molecular cloning. Gene recombination technology was introduced to construct the Trop-2 CAR vector. The expression of Trop-2 CAR on 293T cells was tested by western blot. The effect of Trop-2 CAR-T cells on the proliferation of ovarian cancer cells was evaluated by CCK-8 assay. We found that Trop-2 CAR-T cells prominently inhibited the growth of ovarian cancer cells with Trop-2 antigen expression (P<0.05), moreover, high levels of IFN-γ and IL-2 were detected in supernatant by ELISA (P<0.01). These results suggest that Trop-2 CAR-T cells have the potential to be a clinical treatment for patients with Trop-2 positive ovarian cancer.     

Proliferation inhibition and apoptosis promotion by dual silencing of VEGF and Survivin in human osteosarcoma

Simultaneous silencing of multiple upregulated genes is an attractive and viable treatment strat-egy for many incurable diseases including cancer.Herein we used a dual gene-targeted siRNA conjugate composed of VEGF and Survivin siRNA sequences in the same backbone to inhibit proliferation and angiogenesis in two human osteosarcoma cell lines.We synthesized siRNA sequences targeting the VEGF and Survivin genes individually (VEGF siRNA and Survivin siRNA) or simultaneously (one-chain-double-target siRNA:dual siRNA).VEGF and Survivin mRNA and protein expression levels in human osteosarcoma MG-63 and Saos-2 cells were detected by qRT- PCR and western blot analysis.VEGF and Survivin protein location and expression were evaluated by immunohistochemistry and immunofluorescence staining.MG-63 and Saos-2 cell migration,proliferation,apoptosis,and angiogenesis were detected by scratch test,MTT assay,flow cytometry,and capillary tube assay respectively.The dual siRNA induced similar downregulation of VEGF and Survivin mRNA and protein levels,compared with VEGF siRNA or Survivin siRNA alone.The dual siRNA caused greater suppression of MG-63 and Saos-2 cell migration,proliferation and angiogenesis,and promoted more cell apoptosis than VEGF siRNA or Survivin siRNA alone,suggesting that the effects of the dual siRNA on inhibiting cell proliferation,migration,and angiogenesis and promoting apoptosis were superior to those of the single-target siRNAs.Simultaneous silencing of VEGF and Survivin using the dual siRNA may be an advantageous alternative for the development of therapeutic strategies against human osteosarcoma.     

mad—overexpression down regulates the malignant growth and p53 mediated apoptosis in human hepatocellular carcinoma BEL—7404 cells

Mad protein has been shown as an antagonist of cMyc protein in some cell lines.The effect of Mad protein to the malignant phenotype of human hepatoma BEL-7404 cell line was investigated experimentally.An eukarryotic vector pCDNA Ⅲ containing full ORF fragment of mad cDNA was transfected into targeted cells.Under G418 selection,stable Mad-overexpressed cells were cloned.Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells.DNA synthesis,cell proliferation and anchorage-independent growth in soft-agar of the madtransfected cells were partially inhibited in comparison to control cells.Flos cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase,resulting in the retardation of cell proliferation.RT-PCR detected a marked inhibition of the expression of cdc25A,an important regulator gene of G0/G1 to S phase in cell cycle.It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-74040M1 cells in the absence of serume.Thus,Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL-7404 cells.     

Overexpression of Aurora-A kinase promotes tumor cell proliferation and inhibits apoptosis in esophageal squamous cell carcinoma cell line

Attrora-A kinase, a serine/threonine protein kinase, is a potential oncogene. Amplification and overexpression of Aurora-A have been found in several types of human tumors, including esophageal squamous cell carcinoma （ESCC）. It has been demonstrated that cells overexpressing Attrora-A are more resistant to cisplatin-induced apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. In this report, we showed that overexpression of Attrora-A through stable transfection of pEGFP-Aurora-A in human ESCC KYSE150 cells significantly promoted cell proliferation and inhibited cisplatin- or UV irradiation-induced apoptosis. Cleavages of caspase-3 and poly （ADPribose） polymerase （PARP） in Attrora-A overexpressing cells were substantially reduced after cisplatin or UV treatment. Furthermore, we found that silencing of endogenous Aurora-A kinase with siRNA substantially enhanced sensitivity to cisplatin- or UV-induced apoptosis in human ESCC EC9706 cells. In parallel, overexpression of Aurora-A potently upregulated the expression of Bcl-2. Moreover, the knockdown of Bcl-2 by siRNA abrogated the Aurora-A＇s effect on inhibiting apoptosis. Taken together, these data provide evidence that Aurora-A overexpression promoting cell proliferation and inhibiting apoptosis, suggesting a novel mechanism that is closely related to malignant phenotype and anti-cancer drugs resistance of ESCC cells.     

PKCα translocation from mitochondria to nucleus is closely related to induction of apoptosis in gastric cancer cells

PKCs have been implicated in the regulation of cellular differentiation, proliferation, apoptosis and signal transduction. It was demonstrated in this study that PKCa was located both at mitochondria and in cytosol in gastric cancer cell line BGC-823. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the translocation of PKCa from both mitochondria and cytosol to nucleus as clearly shown by laser-scanning-confocal microscopy, while the protein level of PKCa was not changed by TPA treatment as detected by Western blot. The results also revealed that TPA-induced translocation of PKCa was in close association with apoptosis induction, and such association was further affirmed by other experiments where various apoptotic stimuli and specific inhibitors of PKC were used. Taken together, these findings indicate that translocation of PKCa from both mitochondria and cytosol to nucleus in gastric cancer cell is accompanied by induction of apoptosis, and may imply a new mechanism of th     

Fucoxanthin induces growth arrest and apoptosis in human bladder cancer T24 cells by up-regulation of p21 and down-regulation of mortalin

Fucoxanthin, a natural carotenoid, has been reported to have anti-cancer activity in human colon cancer cells, human prostate cancer cells, human leukemia cells, and human epithelial cervical cancer cells. This study was undertaken to evaluate the molecular mechanisms of fuco- xanthin against human bladder cancer T24 cell line. MTT analysis results showed that 5 and 10 ixM fucoxanthin inhibited the proliferation of T24 cells in a dose- and time- dependent manner accompanied by the growth arrest at Go/G1 phase of cell cycle, which is mediated by the up-regu- lation of p21, a cyclin-dependent kinase （CDK）-inhibitory protein and the down-regulation of CDK-2, CDK-4, cyclin D1, and cyclin E. In addition, 20 and 40 μM fucoxanthin induced apoptosis of T24 cells by the abrogation of morta- lin-p53 complex and the reactivation of nuclear mutant- type p53, which also had tumor suppressor function as wild-type p53. All these results demonstrated that the anti- cancer activity of fucoxanthin on T24 cells was associated with cell cycle arrest at Go/G1 phase by up-regulation of p21 at low doses and apoptosis via decrease in the expres- sion level of mortalin, which is a stress regulator and a mem- ber of heat shock protein 70, followed by up-regulation of cleaved caspase-3 at high doses.     

Ectopic expression of clusterin／apolipoprotein J or Bcl-2 decreases thesensitivity of HaCaT cells to toxic effects of ropivacaine

Local anesthetics inhibit cell proliferation and induce apoptosis in various cell types. Ropivacaine, a unique, novel tertiary amine-type anesthetic, was shown to inhibit the proliferation of several cell types including keratinocytes. We found that Ropivacaine could inhibit the proliferation and induce apoptosis in an immortalized human keratinocyte line,HaCaT, in a dose- and time-dependent manner and with the deprivation of serum. The dose-dependent induction of apoptosis by ropivacaine was demonstrated by DNA fragmentation analysis and the proteolytic cleavage of a caspase-3 substrate—poly (ADP-ribose) polymerase (PARP). In addition, ropivacaine downregulated the expression of clusterin/ apoliporotein J, a protein with anti-apoptotic properties, in a dose-dependent manner, which well correlated with the induction of apoptosis of HaCaT cells. To investigate the role of clusterin/apoliporotein J in ropivacaine-induced apoptosis,HaCaT cells overexpressing clusterin/apoliporotein J were generated and compared to cells expressing the well established anti-apoptotic Bcl-2 protein. Ectopic overexpression of the secreted form of clusterin/apoliporotein J or Bcl-2decreased the sensitivity of HaCaT cells to toxic effects of ropivacaine as demonstrated by DNA fragmentation, the proteolytic cleavage of PARP and by a reduction in procaspase-3 expression. Furthermore, the downregulation of endogenous clusterin/apolipoprotein J levels by ropivacaine suggested that this might be one mechanism by which ropivacaine induced cell death in HaCaT cells. In conclusion, the ability of ropivacaine to induce antiproliferative responses and to suppress the expression of the anti-apoptotic protein clusterin/apolipoprotein J, combined with previously reported anti-inflammatory activity and analgesic property of the drug, suggests that ropivacaine may have potential utility in the local treatment of tumors.     

PKCa translocation from mitochondria to nucleus is closely related to induction of apoptosis in gastric cancer cells

PKCs have been implicated in the regulation of cellular differentiation, proliferation, apoptosis and signal transduction. It was demonstrated in this study that PKCα was located both at mitochondria and in cytosol in gastric cancer cell line BGC-823. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the translocation of PKCα from both mitochondria and cytosol to nucleus as clearly shown by laser-scanning-confocal microscopy, while the protein level of PKCα was not changed by TPA treatment as detected by Western blot. The results also revealed that TPA-induced translocation of PKCα was in close association with apoptosis induction, and such association was further affirmed by other experiments where various apoptotic stimuli and specific inhibitors of PKC were used. Taken together, these findings indicate that translocation of PKCα from both mitochondria and cytosol to nucleus in gastric cancer cell is accompanied by induction of apoptosis, and may imply a new mechanism of the potential linking between cell apoptosis and PKCα translocation.     

Down-regulation of GPR137 expression inhibits proliferation of colon cancer cells

G protein-coupled receptors （GPRs） are highly related to oncogenesis and cancer metastasis. G protein-coupled re- ceptor 137 （GPR137） was initially reported as a novel orphan GPR about 10 years ago. Some orphan GPRs have been implicated in human cancers. The aim of this study is to investigate the role of GPR137 in human colon cancer. Expression levels of GRP137 were analyzed in different colon cancer cell lines by quantitative polymerase chain re- action and western blot analysis. Lentivirus-mediated short hairpin RNA was specifically designed to knock down GPR137 expression in colon cancer cells. Cell viability was measured by methylthiazoletetrazolium and colony forma- tion assays. In addition, cell cycle characteristic was investi- gated by flow cytometry. GRP137 expression was observed in aH seven colon cancer cell lines at different levels. The mRNA and protein levels of GPR137 were down-regulated in both HCTll6 and RKO cells after lentivirus infection. Lentivirus-mediated silencing of GPR137 reduced the proliferation rate and colonies numbers. Knockdown of GPR137 in both cell lines led to cell cycle arrest in the G0/G1 phase. These results indicated that GPR137 plays an important role in colon cancer cell proliferation. A better understanding of GPR137＇s effects on signal transduction pathways in colon cancer cells may provide insights into the novel gene therapy of colon cancer.     

miR-195-5p exerts tumor-suppressive functions in human lung cancer cells through targeting TrxR2

miR-195-5p has been widely explored in various cancers and is considered as a tumor-suppressive microRNA.However,its roles in human lung cancer pathogenesis are not fully elucidated.In this study,we aimed to explore how miR-195-5p is involved in malignant behaviors of lung adenocarcinoma(LUAD)cells.miR-195-5p expression was examined in the tumor tissues of patients with LUAD and human LUAD cell lines including A549 and PC-9.Thioredoxin reductase 2(TrxR2)was predicted to be an mRNA target of miR-195-5p using online tools and validated by the Dual-Luciferase Reporter Assay.Lentivirus infection was used for gene overexpression,while gene knockdown was achieved by RNA interference.Cell proliferation was determined by Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine methods,and cell migration and invasion were assayed with transwell experiments.Cell apoptosis was determined by annexin V staining-based flow cytometry.The antitumor effects of miR-195-5p were also evaluated in nude mice xenografted with A549 cells.We found that miR-195-5p was lowly expressed in human LUAD cells,and its overexpression markedly suppressed cell proliferation,migration,and invasion and increased the apoptosis of LUAD cells in vitro.TrxR2 knockdown phenocopied the tumor-suppressive effects of miR-195-5p overexpression,while simultaneous TrxR2 overexpression remarkably reversed the effects of miR-195-5p overexpression on malignant behaviors of A549 and PC-9 cells.Additionally,miR-195-5p overexpression inhibited the growth of xenografted A549 tumor in nude mice.Our work verified that miR-195-5p exerts tumor-suppressive functions in LUAD cells through targeting TrxR2 and suggested that the miR-195-5p/TrxR2 axis is a potential biomarker for LUAD therapy.     

Overexpression of PTEN induces cell growth arrest and apoptosis in human breast cancer ZR-75-1 cells

Phosphatase and tensin homolog （PTEN） is a tumor suppressor gene located at human chromosome 10q23, might play an important role in cell proliferation, cell cycle and apoptosis of cancer cells. In this study, the eukaryotic expression vectors pBP-wt-PTEN （containing a wild-type PTEN gene） and pBP-G129R-PTEN （containing a mutant PTEN gene） were used to transfect breast cancer ZR-75-1 cells. After transfection, ZR-75-1 cells expressing PTEN were obtained and tested. The blue exclusion assay showed the growth rate of the cells transfected with pBP-wt-PTEN was significantly lower than that of the control cells transfected with pBP-G129R-PTEN. Analysis of the cell cycle by flow cytometry showed that the progression from the G1 to the S phase was arrested in cells expressing wild-type PTEN. Some typical morphological changes of apoptosis were also observed in cells transfected with pBP-wt-PTEN, but not in those transfected with pBP-G 129R-PTEN. This study shows that overexpression of PTEN in ZR-75-1 cells leads to cell growth arrest and apoptosis.     

Mechanism of taurine-induced apoptosis in human colon cancer cells

Taurine （Tau） has been shown to possess cancer therapeutic effect through induction of apoptosis, while the underlying molecular mechanism of its anti-cancer effect is not well understood. PUMA （p53-upregulated modulator of apoptosis） plays an important role in the process of apoptosis induction in a variety of human tumor ceils in both p53- dependent and -independent manners. However, whether PUMA is involved in the process of Tau-induced apoptosis in cancer cells has not been well studied. In the present study, we treated human colorectal cancer cells HT-29 （mutant p53） and LoVo （wild-type p53） with different concentrations of Tau, which led to the repression of cell proliferation and induction of apoptosis in both cell fines. Meanwhile, we also observed the increased expression of PUMA and high Bax/Bcl-2 ratios. To determine the role of PUMA in Tau-induced apoptosis, we used small interfering RNA interference to suppress PUMA expression. As a result, apoptosis was decreased in response to Tau treatment. All these results indicated that PUMA plays a critical role in Tauinduced apoptosis pathway in human colorectal cancer ceils. Demonstration of the molecular mechanism involved in the anti-tumor effect of Tau may be useful in the therapeutic target selection for p53-deficient colorectal cancer.