Triptolide induces pancreatic cancer cell death via inhibition of heat shock protein 70

Pancreatic cancer is highly resistant to current chemotherapy agents. We therefore examined the effects of triptolide (a diterpenoid triepoxide) on pancreatic cancer growth and local-regional tumor spread using an orthotopic model of pancreatic cancer. We have recently shown that an increased level of HSP70 in pancreatic cancer cells confers resistance to apoptosis and that inhibiting HSP70 induces apoptosis in these cells. In addition, triptolide was recently identified as part of a small molecule screen, as a regulator of the human heat shock response. Therefore, our aims were to examine the effects of triptolide on (a) pancreatic cancer cells by assessing viability and apoptosis, (b) pancreatic cancer growth and local invasion in vivo, and (c) HSP70 levels in pancreatic cancer cells. Incubation of PANC-1 and MiaPaCa-2 cells with triptolide (50-200 nmol/L) significantly reduced cell viability, but had no effect on the viability of normal pancreatic ductal cells. Triptolide induced apoptosis (assessed by Annexin V, caspase-3, and terminal nucleotidyl transferase-mediated nick end labeling) and decreased HSP70 mRNA and protein levels in both cell lines. Triptolide (0.2 mg/kg/d for 60 days) administered in vivo decreased pancreatic cancer growth and significantly decreased local-regional tumor spread. The control group of mice had extensive local invasion into adjacent organs, including the spleen, liver, kidney, and small intestine. Triptolide causes pancreatic cancer cell death in vitro and in vivo by induction of apoptosis and its mechanism of action is mediated via the inhibition of HSP70. Triptolide is a potential therapeutic agent that can be used to prevent the progression and metastases of pancreatic cancer.     

Synergism of Cytotoxicity Effects of Triptolide and Artesunate Combination Treatment in Pancreatic Cancer Cell Lines

Background: Triptolide, extracted from the herb Tripteryglum wilfordii Hook.f that has long been used as anatural medicine in China, has attracted much interest for its anti-cancer effects against some kinds of tumoursin recent years. Artesunate, extracted from the Chinese herb Artemisia annua, has proven to be effective andsafe as an anti-malarial drug that possesses anticancer potential. The present study attempted to clarify iftriptolide enhances artesunate-induced cytotoxicity in pancreatic cancer cell lines in vitro and in vivo. Methods:In vitro, to test synergic actions, cell viability and apoptosis were analyzed after treatment of pancreatic cancercell lines with the two agents singly or in combination. The molecular mechanisms of apoptotic effects were alsoexplored using qRT-PCR and Western blotting. In vivo, a tumor xenograft model was established in nude mice,for assessment of inhibitory effects of triptolide and artesunate. Results: We could show that the combinationof triptolide and artesunate could inhibit pancreatic cancer cell line growth, and induce apoptosis, accompaniedby expression of HSP 20 and HSP 27, indicating important roles in the synergic effects. Moreover, tumor growthwas decreased with triptolide and artesunate synergy. Conclusion: Our result indicated that triptolide andartesunate in combination at low concentrations can exert synergistic anti-tumor effects in pancreatic cancercells with potential clinical applications.     

Enhanced antitumor effect of combined triptolide and ionizing radiation.

PURPOSE: The lack of effective treatment for pancreatic cancer results in a very low survival rate. This study explores the enhancement of the therapeutic effect on human pancreatic cancer via the combination of triptolide and ionizing radiation (IR). EXPERIMENTAL DESIGN: In vitro AsPC-1 human pancreatic cancer cells were treated with triptolide alone, IR alone, or triptolide plus IR. Cell proliferation was analyzed with sulforhodamine B (SRB) method and clonogenic survival; comparison of apoptosis induced by the above treatment was analyzed by annexin V-propidium iodide (PI) staining. Furthermore, the expression of apoptotic pathway intermediates was measured by the assay of caspase activity and Western blot. Mitochondrial transmembrane potential was determined by JC-1 assay. In vivo, AsPC-1 xenografts were treated with 0.25 mg/kg triptolide, 10 Gy IR, or triptolide plus IR. The tumors were measured for volume and weight at the end of the experiment. Tumor tissues were tested for terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) and immunohistochemistry. RESULTS: The combination of triptolide plus IR reduced cell survival to 21% and enhanced apoptosis, compared with single treatment. In vivo, tumor growth of AsPC-1 xenografts was reduced further in the group treated with triptolide plus IR compared with single treatment. TUNEL and immunohistochemistry of caspase-3 cleavage in tumor tissues indicated that the combination of triptolide plus IR resulted in significantly enhanced apoptosis compared with single treatments. CONCLUSIONS: Triptolide in combination with ionizing radiation produced synergistic antitumor effects on pancreatic cancer both in vitro and in vivo and seems promising in the combined modality therapy of pancreatic cancer.     

HSP70反义寡脱氧核苷酸增强宫颈癌和卵巢癌细胞的热敏感性

观察ＨＳＰ７０反义寡脱氧核苷酸对宫颈癌细胞和卵巢癌细胞热敏感性的影响。方法计数集落形成率,了解宫颈癌细胞和卵巢癌细胞的热敏感性。用光镜,琼脂糖凝胶电泳及流式细胞术观察凋亡细胞。结果经ＨＳＰ７０反义寡脱氧核苷酸预先处理的细胞增强了对热处理的敏感性,热敏感性的增加与ＨＳＰ７０反义寡脱氧核苷酸的处理剂量相关。用形态学、琼脂糖凝胶电泳及流式细胞术均证经实ＨＳＰ７０反义寡脱氧核苷酸及热处理的细胞发生了凋亡。     

Increased expression of phospho-acetyl-CoA carboxylase protein is an independent prognostic factor for human gastric cancer without lymph node metastasis

Triptolide is a traditional Chinese medicinal herb-derived antineoplastic agent. However, its antitumor activity against gynecologic carcinomas has not yet been well described. It is the purpose of this article to investigate the effect and mechanism of triptolide in human ovarian cancer using both A2780 (p53 wild) and OVCAR-3 (p53 mutated) cells. Our results showed that triptolide exerted a potent inhibitory effect on the growth and proliferation of both cell lines in a dose- and time-dependent manner and that the effect was independent of the expression of p53. In contrast, triptolide had only a marginal cytotoxicity in noncancerous ovary cells, lung fibroblast cells, and macrophage cells, indicating differential inhibitory effects of the drug on cell growth between ovarian cancer cells and normal tissue cells. Exposure of the ovarian cancer cells to triptolide induced apoptosis, as evaluated by annexin V/propidium iodide-labeled flow cytometry. Triptolide-induced apoptosis was accompanied by cytochrome c release and caspase-3 activation and was associated with downregulation of Bcl-2 and upregulation of Bax. Cell cycle analysis demonstrated that treatment with triptolide induced cell cycle S phase arrest in A2780 cells and G2/M phase arrest in OVCAR-3 cells. Further detection by Western blotting revealed that the cell cycle arrest by triptolide in both cell lines occurred in concert with increased expression of p21^CIP1/WAF1. This study shows that triptolide selectively kills ovarian cancer cells with different p53 status predominantly through regulating the coordinate and dynamic cellular processes of proliferation and apoptosis, thereby making it a promising chemotherapeutic agent against a broad spectrum of ovarian carcinomas.     

Sphingosine kinase-1 inhibition sensitizes curcumin-induced growth inhibition and apoptosis in ovarian cancer cells

Recent published studies suggest that increasing levels of ceramides enhance the chemo-sensitivity of curcumin. Using in vitro approaches, we analyzed the impact of sphingosine kinase-1 (SphK-1) inhibition on ceramide production, and evaluated SphK1 inhibitor II (SKI-II) as a potential curcumin chemo-sensitizer in ovarian cancer cells. We found that SphK1 is overexpressed in ovarian cancer patients' tumor tissues and in cultured ovarian cancer cell lines. Inhibition of SphK1 by SKI-II or by RNA interference (RNAi) knockdown dramatically enhanced curcumin-induced apoptosis and growth inhibition in ovarian cancer cells. SKI-II facilitated curcumin-induced ceramide production, p38 activation and Akt inhibition. Inhibition of p38 by the pharmacological inhibitor (SB 203580), a dominant-negative expression vector, or by RNAi diminished curcumin and SKI-II co-administration-induced ovarian cancer cell apoptosis. In addition, restoring Akt activation introducing a constitutively active Akt, or inhibiting ceramide production by fumonisin B1 also inhibited the curcumin plus SKI-II co-administration-induced in vitro anti-ovarian cancer effect, suggesting that ceramide accumulation, p38 activation and Akt inhibition are downstream effectors. Our findings suggest that low, well-tolerated doses of SKI-II may offer significant improvement to the clinical curcumin treatment of ovarian cancer.     

NSC606985 induces apoptosis,exerts synergistic effects with cisplatin,and inhibits hypoxia-stabilized HIF-1α protein in human ovarian cancer cells

The camptothecins, which target the intranuclear enzyme topoisomerase I, have advanced to the forefront of several areas of developmental chemotherapy of cancers. In the present study, we investigated the potential anti-human ovarian cancer effects of NSC606985, a novel and rarely studied camptothecin analog, and its combination with cisplatin (CDDP). Human ovarian cancer cell line COC1 cells were treated with different nanomolar of NSC606985 with or without CDDP, and cell growth and apoptosis were evaluated, respectively, by MTT assay and annexin-V assay on flow cytometry. Chou–Talalay analysis was used to evaluate combined effect of NSC606985 and CDDP. Western blot was used to detect protein kinase Cδ (PKCδ), caspase-3 and hypoxia-inducible factor-1α (HIF-1α) proteins. Our results showed that NSC606985 at nanomolar concentration induced apoptosis with the activation of PKCδ in COC1 cells. Especially, NSC606985 presented the significant combined effects on COC1 cells in terms of growth inhibition and apoptosis induction. In addition, NSC606985 significantly antagonized the accumulation of HIF-1α stabilized by hypoxia or hypoxia-mimetic agent. These results suggest that NSC606985 and its combination with CDDP present the therapeutic potential on ovarian cancer, and deserve further preclinical and clinical studies.     

The 70kilodalton heat shock protein is an inhibitor of apoptosis in prostate cancer

The 70?kD heat shock protein (HSP70) plays essential cellular roles in mediating intra-cellular protein folding and protecting cells from proteotoxic stress. This study has examined the role of HSP70 in the expression of apoptosis in prostate carcinoma cells. Apoptosis was negatively correlated with HSP70 expression in PC-3 cells heat shocked in vivo. Further experiments carried out on an in vitro reconstituted system with isolated nuclei and cytoplasm from PC-3 cells showed that purified HSP70 directly inhibits apoptosis in a dose-dependant manner. Therefore, the potential role of depletion of intracellular HSP70 was examined as a means of inducing apoptosis in PC-3 cancer cells. Depletion of HSP70 by two independent strategies, either with anti-sense oligonucleotides directed against HSP70 mRNA or with the bioflavinoid drug quercetin, led to apoptosis in the absence of stress. In addition, quercetin pre-treatment synergistically enhanced apoptosis in combination with heat shock. Thus, HSP70 plays a physiological role in tumour cells as an inhibitor of apoptosis occurring both spontaneously and after stress and is a potential target for apoptosis-based cancer therapy.     

The 70 kilodalton heat shock protein is an inhibitor of apoptosis in prostate cancer.

The 70 kD heat shock protein (HSP70) plays essential cellular roles in mediating intracellular protein folding and protecting cells from proteotoxic stress. This study has examined the role of HSP70 in the expression of apoptosis in prostate carcinoma cells. Apoptosis was negatively correlated with HSP70 expression in PC-3 cells heat shocked in vivo. Further experiments carried out on an in vitro reconstituted system with isolated nuclei and cytoplasm from PC-3 cells showed that purified HSP70 directly inhibits apoptosis in a dose-dependant manner. Therefore, the potential role of depletion of intracellular HSP70 was examined as a means of inducing apoptosis in PC-3 cancer cells. Depletion of HSP70 by two independent strategies, either with anti-sense oligonucleotides directed against HSP70 mRNA or with the bioflavinoid drug quercetin, led to apoptosis in the absence of stress. In addition, quercetin pre-treatment synergistically enhanced apoptosis in combination with heat shock. Thus, HSP70 plays a physiological role in tumour cells as an inhibitor of apoptosis occurring both spontaneously and after stress and is a potential target for apoptosis-based cancer therapy.     

Dual targeting of HSC70 and HSP72 inhibits HSP90 function and induces tumor-specific apoptosis

Heat-shock protein 70 (HSP70) isoforms contribute to tumorigenesis through their well-documented antiapoptotic activity and via their role as cochaperones for the HSP90 molecular chaperone. HSP70 expression is induced following treatment with HSP90 inhibitors, which may attenuate the cell death effects of this class of inhibitor. Here we show that silencing either heat-shock cognate 70 (HSC70) or HSP72 expression in human cancer cell lines has no effect on HSP90 activity or cell proliferation. However, simultaneously reducing the expression of both of these isoforms induces proteasome-dependent degradation of HSP90 client proteins, G1 cell-cycle arrest, and extensive tumor-specific apoptosis. Importantly, simultaneous silencing of HSP70 isoforms in nontumorigenic cell lines does not result in comparable growth arrest or induction of apoptosis, indicating a potential therapeutic window.     

Induction of MicroRNA-9 Mediates Cytotoxicity of Curcumin Against SKOV3 Ovarian Cancer Cells

Background: Curcumin, a phenolic compound extracted from the rhizomes of Curcuma longa, has showncytotoxic effects against a variety of cancers. The aim of this study was to identify potential microRNA (miRNA)mediators of the anticancer effects of curcumin in ovarian cancer cells. Materials and Methods: SKOV3 ovariancancer cells were treated with curcumin (10-60 μM) and miR-9 expression, cell proliferation, and apoptosiswere assessed. The effects of miR-9 depletion on curcumin-mediated growth suppression were also examined.Phosphorylation of Akt and forkhead box protein O1 (FOXO1) was measured in cells with miR-9 overexpressionor curcumin treatment. Results: Curcumin caused a significant and dose-dependent increase of miR-9 expressionin SKOV3 cells, while significantly impeding cell proliferation and stimulating apoptosis. Depletion of miR-9significantly (p<0.05) attenuated the growth-suppressive effects of curcumin on SKOV3 cells, coupled withreduced percentages of apoptotic cells. In contrast, overexpression of miR-9 significantly enhanced the cleavageof caspase-3 and poly(ADP-ribose) polymerase and promoted apoptotic death in SKOV3 cells. Western blotanalysis showed that both miR-9 overexpression and curcumin similarly caused a significant (p<0.05) declinein the phosphorylation of Akt and FOXO1, compared to untreated cells. Conclusions: The present studyprovided evidence that curcumin exerts its cytotoxic effects against SKOV3 ovarian cancer cells largely throughupregulation of miR-9 and subsequent modulation of Akt/FOXO1 axis. Further studies are needed to identifydirect targets of miR-9 that mediate the anticancer effects of curcumin in ovarian cancer cells.     

Arsenic trioxide and cisplatin synergism increase cytotoxicity in human ovarian cancer cells: Therapeutic potential for ovarian cancer

Drug resistance is a major concern in the successful treatment of ovarian cancer. In the present study we report a combinational drug regime using arsenic trioxide (ATO) and cisplatin (CDDP) to increase therapeutic potentiality in ovarian cancer cells. ATO-mediated growth inhibition and apoptosis in human suspension ovarian cancer COC1 cells were evaluated by MTT assay and annexin V assay using flow cytometry, respectively. cDNA arrays were performed to screen ATO-mediated gene expression. Treatment of COC1 cells with ATO alone resulted in growth inhibition and apoptosis with a dose-and time-dependent fashion; further cDNA arrays showed that 34 genes (23 up-regulated genes and 11 down-regulated genes) may strongly associate with the antiproliferative and pro-apoptotic effects induced by ATO. Furthermore, Chou–Talalay analysis was used to evaluate the combinational effect of ATO and CDDP as well as dose-reduction index (DRI) in a panel of ovarian cancer cells including CDDP-sensitive and -resistant cell lines. The combination index (CI) analysis indicated that the interaction effect of ATO/CDDP exhibited a wide range of synergism in all the adherent ovarian cancer cells (A2780, IGROV-1, SKOV-3, and R182) as well as 0.93 to 0.69 for IC₅₀ to IC₉₀ in suspension COC1 cells where CI < 1, =1, and >1, define synergism, additive effect, and antagonism, respectively. More intriguingly, the combination of ATO and CDDP yielded favorable DRIs ranging from 1.23-fold to 13.51-fold dose reduction. These results suggest that ATO and its combination with CDDP present therapeutic potential for ovarian cancer, and deserve further preclinical and clinical studies. ( Cancer Sci 2009; 100: 2459–2464)     

Photosensitizer effect of curcumin on UVB-irradiated HaCaT cells through activation of caspase pathways

Photodynamic therapy (PDT) using photosensitizer can induce diverse cellular responses, including apoptosis. Recently, it has been reported that PDT using methylaminolaevulinate may be effective in basal cell carcinoma. However, it is largely unknown whether PDT using a natural product such as curcumin can induce apoptosis in skin cancer. In this study, to confirm the photodynamic effect of curcumin, we investigated the synergistic effect of the combination of UVB with curcumin on apoptotic cell death in HaCaT cells and molecular mechanisms underlying apoptosis. Our data showed that HaCaT cells were markedly undergoing apoptosis, evidenced by DNA laddering, by combination of UVB with curcumin, compared to UVB or curcumin alone. Furthermore, combination of UVB irradiation with curcumin synergistically induces apoptotic cell death in HaCaT cells through activation of caspase-8, and -3 as well as caspase-9 activation followed by release of cytochrome c. Thus, our data indicate that curcumin may be a promising photosensitizer used in PDT to induce apoptosis in skin cancer cells.     

Triptolide induces apoptosis in human adrenal cancer NCI-H295 cells through a mitochondrial-dependent pathway

Triptolide, the main active component obtained from Tripterygium wilfordii Hook. f, has been reported to present potent immunosuppressive and anti-inflammatory biological activities. It has been previously shown that due to the cytotoxicity of triptolide it has a limited use in the clinic. Although numerous studies have shown that triptolide induced apoptosis in many human cancer cells there is no report to show triptolide-induced apoptosis in human adrenal cancer cells. We treated the human adrenal cancer NCI-H295 cells with triptolide in vitro and investigated its cytotoxic effects. The cytotoxicity was examined and quantitated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and the viability of inhibition and apoptosis was determined by flow cytometric assay, using propidium iodide (PI) staining for apoptosis. Flow cytometric assay also used for the determination of reactive oxygen species (ROS) production and the levels of mitochondrial membrane potential (ΔΨm), and the caspase-3 and -9 activation in NCI-H295 cells. Western blotting was used for examining the changes of apoptotic associated proteins. The results indicated that triptolide induced cytotoxicity (decreased the percentage of viable cells) and induced sub-G1 phase (apoptosis) occurring in NCI-H295 cells and those effects are dose-dependent. Results also showed that triptolide promoted the production of ROS and decreased the levels of ΔΨm in examined NCI-H295 cells. The results showed that triptolide promoted the levels of cytochrome c, Apaf-1, AIF, Endo G, caspase-9 and -3 which were analyzed by Western blotting. These results suggest that triptolide is able to induce apoptosis on NCI-H295 cells through the mitochondrial-dependent signal pathway.     

Heat shock protein 70 (HSP70) does not prevent the inhibition of cell growth in DU-145 cells treated with TGF-beta1

BACKGROUND: Mitogen-activated protein kinase (MAPK) is one of the transforming growth factor-beta (TGF-beta signaling pathways while heat shock protein 70 (HSP70) prevents apoptosis by affecting MAPK signaling downstream. However, the interrelationship between TGF-beta and HSP70 signaling is still unknown. MATERIALS AND METHODS: DU-145 prostate cancer cells were treated with 40 pM and 200 pM TGF-beta1. After 3, 6, 9, 12 and 24 hours, cell proliferation assay and cell cycle analysis were performed. The activities of HSP70 and MAPKs (c-Jun N-terminal kinase 1 (JNK1), extracellular signal-regulated kinase 1 (ERK1), ERK2 and p38) were analyzed by Western blot at each time-point. RESULTS: TGF-beta1 inhibited the cell growth in a dose-dependent manner at 3 hours. Late G1 accumulation in the cell cycle was observed in a dose-dependent manner after 24 hours. HSP70 and JNK1 increased only at 3 hours and decreased for up to 24 hours thereafter. ERK1, ERK2 and p38 decreased from 3 to 24 hours after TGF-beta1 treatment. CONCLUSION: These data suggest that HSP70 does not prevent the inhibition of cell growth in DU-145 cells treated with TGF-beta1.     

多烯紫杉醇和姜黄素联用对人肺腺癌A549细胞增殖和凋亡的影响

目的研究姜黄素和多烯紫杉醇对肺腺癌A549细胞增殖及调亡的影响,并探讨两种药物的合用是否可增强抗癌作用及降低不良反应。方法体外培养肺腺癌细胞系A549细胞,采用 MTT 法计算细胞抑制率;Giemsa 染色法观察细胞形态学变化;流式细胞仪分析细胞的凋亡率。结果经MTT法检测显示,姜黄素能明显抑制A549细胞的生长,多烯紫杉醇50 μg/ml与姜黄素5、10、20 μmol/L联用对A549细胞的抑制作用明显大于两药单用时的抑制作用（P<0.01）;Giemsa染色显示多烯紫杉醇与姜黄素联用后诱导细胞凋亡的作用增强,可见典型的凋亡小体;流式细胞仪结果表明姜黄素能增加多烯紫杉醇的细胞凋亡率,其作用呈剂量依赖性。结论姜黄素与多烯紫杉醇可协同抑制人肺腺癌A549细胞的增殖;二者联合化疗有增效减毒作用。     

Synthetic small interfering RNA targeting heat shock protein 105 induces apoptosis of various cancer cells both in vitro and in vivo

We previously reported that heat shock protein 105 (HSP105), identified by serological analysis of a recombinant cDNA expression library (SEREX) using serum from a pancreatic cancer patient, was overexpressed in various human tumors and in the testis of adult men by immunohistochemical analysis. In the present study, to elucidate the biological function of the HSP105 protein in cancer cells, we first established NIH3T3 cells overexpressing murine HSP105 (NIH3T3-HSP105). The NIH3T3-HSP105 cells acquired resistance to apoptosis induced by heat shock or doxorubicin. The small interfering RNA (siRNA)-mediated suppression of HSP105 protein expression induced apoptosis in human cancer cells but not in fibroblasts. By a combination of siRNA introduction and doxorubicin or heat shock treatment, apoptosis was induced synergistically in a human colon cancer cell line, HCT116. In vivo , siRNA inoculation into the human gastric cancer cell line KATO-3 established in the flank of an NOD SCID mouse suppressed the tumor growth. This siRNA-induced apoptosis was mediated through caspases, but not the p53 tumor suppressor protein, even though the HSP105 protein was bound to wild-type p53 protein in HCT116 cells. These findings suggest that the constitutive overexpression of HSP105 in cancer cells is involved in malignant transformation by protecting tumor cells from apoptosis. HSP105 may thus be a novel target molecule for cancer therapy and a treatment regimen using synthetic siRNA to suppress the expression of HSP105 protein may provide a new strategy for cancer therapy. ( Cancer Sci 2006; 97: 623–632)     

Co-inhibition of HSP70/HSP90 synergistically sensitizes nasopharyngeal carcinoma cells to thermotherapy

The upregulation of both HSP70 and HSP90 frequently compromises the effects of thermotherapy. The co-inhibition of HSP70/HSP90 may be preferable to enhance the effects of thermotherapy on nasopharyngeal carcinoma cells. The changes of HSP70 and HSP90 were detected after thermotherapy in human nasopharyngeal cancer cell HNE1. 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) and quercetin were used to inhibit the activity of HSP90 and HSP70. The enhanced effects were evaluated in vitro and in vivo. Both HSP70 and HSP90 were upregulated promptly in HNE1 after thermotherapy. Single inhibition of HSP70 resulted in overexpression and delayed descent of HSP90. The co-inhibition of HSP70/HSP90 with quercetin plus 17-DMAG significantly increased apoptosis in hyperthermia-treated HNE1 cells both in vitro and in vivo. The co-inhibition of HSP70/HSP90 synergistically sensitizes nasopharyngeal carcinoma cells to hyperthermia.