A Long Non-Coding RNA snaR Contributes to 5-Fluorouracil Resistance in Human Colon Cancer Cells

Several types of genetic and epigenetic regulation have been implicated in the development of drug resistance, one significant challenge for cancer therapy. Although changes in the expression of non-coding RNA are also responsible for drug resistance, the specific identities and roles of them remain to be elucidated. Long non-coding RNAs (lncRNAs) are a type of ncRNA (> 200 nt) that influence the regulation of gene expression in various ways. In this study, we aimed to identify differentially expressed lncRNAs in 5-fluorouracil-resistant colon cancer cells. Using two pairs of 5-FU-resistant cells derived from the human colon cancer cell lines SNU-C4 and SNU-C5, we analyzed the expression of 90 lncRNAs by qPCR-based profiling and found that 19 and 23 lncRNAs were differentially expressed in SNU-C4R and SNU-C5R cells, respectively. We confirmed that snaR and BACE1AS were downregulated in resistant cells. To further investigate the effects of snaR on cell growth, cell viability and cell cycle were analyzed after transfection of siRNAs targeting snaR. Down-regulation of snaR decreased cell death after 5-FU treatment, which indicates that snaR loss decreases in vitro sensitivity to 5-FU. Our results provide an important insight into the involvement of lncRNAs in 5-FU resistance in colon cancer cells.     

Reduction of Orc6 Expression Sensitizes Human Colon Cancer Cells to 5-Fluorouracil and Cisplatin

Previous studies from our group have shown that the expression levels of Orc6 were highly elevated in colorectal cancer patient specimens and the induction of Orc6 was associated with 5-fluorouracil (5-FU) treatment. The goal of this study was to investigate the molecular and cellular impact of Orc6 in colon cancer. In this study, we use HCT116 (wt-p53) and HCT116 (null-p53) colon cancer cell lines as a model system to investigate the impact of Orc6 on cell proliferation, chemosensitivity and pathways involved with Orc6. We demonstrated that the down regulation of Orc6 sensitizes colon cancer cells to both 5-FU and cisplatin (cis-pt) treatment. Decreased Orc6 expression in HCT-116 (wt-p53) cells by RNA interference triggered cell cycle arrest at G1 phase. Prolonged inhibition of Orc6 expression resulted in multinucleated cells in HCT-116 (wt-p53) cell line. Western immunoblot analysis showed that down regulation of Orc6 induced p21 expression in HCT-116 (wt-p53) cells. The induction of p21 was mediated by increased level of phosphorylated p53 at ser-15. By contrast, there is no elevated expression of p21 in HCT-116 (null-p53) cells. Orc6 down regulation also increased the expression of DNA damaging repair protein GADD45β and reduced the expression level of JNK1. Orc6 may be a potential novel target for future anti cancer therapeutic development in colon cancer.     

MicroRNA Profiling in Human Colon Cancer Cells during 5-Fluorouracil-Induced Autophagy

Autophagy modulation is now recognized as a potential therapeutic approach for cancer (including colorectal cancer), yet the molecular mechanisms regulating autophagy in response to cellular stress are still not well understood. MicroRNAs (miRNAs) have been found to play important roles in controlling many cellular functions, including growth, metabolism and stress response. The physiological importance of the miRNA-autophagy interconnection is only beginning to be elucidated. MiRNA microarray technology facilitates analysis of global miRNA expression in certain situations. In this study, we explored the expression profile of miRNAs during the response of human colon cancer cells (HT29s) to 5-FU treatment and nutrient starvation using miRNA microarray analysis. The alteration of miRNA expression showed the same pattern under both conditions was further testified by qRT-PCR in three human colon cancer cell lines. In addition, bioinformatic prediction of target genes, pathway analysis and gene network analysis were performed to better understand the roles of these miRNAs in the regulation of autophagy. We identified and selected four downregulated miRNAs including hsa-miR-302a-3p and 27 upregulated miRNAs under these two conditions as having the potential to target genes involved in the regulation of autophagy in human colon cancer cells. They have the potential to modulate autophagy in 5-FU-based chemotherapy in colorectal cancer.     

Inhibition of Transient Receptor Potential Channel 5 Reverses 5-Fluorouracil Resistance in Human Colorectal Cancer Cells

5-Fluorouracil (5-Fu) is commonly used in the chemotherapy of colorectal cancer (CRC), but resistance to 5-Fu occurs in most cases, allowing cancer progression. Suppressing ABCB1 (ATP-binding cassette, subfamily B, member 1), which is a pump overproduced in cancer cells to export cytotoxic drugs, is an attractive strategy to overcome drug resistance. In the present study, transient receptor potential channel TrpC5 was found to be overproduced at the mRNA and protein levels together with ABCB1 in 5-Fu-resistant human CRC HCT-8 (HCT-8/5-Fu) and LoVo (LoVo/5-Fu) cells. More nuclear-stabilized β-catenin accumulation was found in HCT-8/5-Fu and LoVo/5-Fu cells than in HCT-8 and LoVo cells. Suppressing TrpC5 expression with TrpC5-specific siRNA inhibited the canonical Wnt/β-catenin signal pathway, reduced the induction of ABCB1, weakened the ABCB1 efflux pump, and caused a remarkable reversal of 5-Fu resistance in HCT-8/5-Fu and LoVo/5-Fu cells. On the contrary, enforcing TrpC5 expression resulted in an activated Wnt/β-catenin signal pathway and up-regulation of ABCB1. Taken together, we demonstrated an essential role of TrpC5 in ABCB1 induction and drug resistance in human CRC cells via promoting nuclear β-catenin accumulation.     

Feline Herpesvirus Infection in Fused Cultures of Naturally Resistant Human Cells

Feline herpesvirus (FHV) attaches to, but does not penetrate, naturally resistant human embryonic lung cells. When the cells with attached virus are subsequently fused with inactivated Sendai virus, FHV causes characteristic cytopathic effects, but no infectious virus can be recovered from the infected cells.     

PXR基因外显子3甲基化与肠癌细胞对5氟尿嘧啶的耐药性相关

孕烷X受体（pregnane X receptor, PXR）可通过调节细胞色素P450同工酶3A4 -CYP3A4的表达而影响肿瘤细胞对化疗的敏感性,而其表达水平则会受到自身基因 甲基化的影响.本文研究了结肠癌组织中pxr基因甲基化的分布情况及其对pxr, cyp3a4表达的影响,并在多种结肠癌细胞系中分析了pxr基因甲基化是否与5-氟尿嘧 啶 (5-FU)耐药性相关.收集结肠癌病灶区、癌旁区及正常结肠组织样本,分别提取基因组DNA及RNA.PCR限制性酶切分析检测pxr基因外显子3甲基化;real-time PCR检测pxr及cyp3a4基因的表达.鉴定LOVO、LS180、LS174T、HT29、HCT116等5种结 肠癌细胞中pxr外显子3甲基化与pxr, cyp3a4表达的相关性并分别筛选出PXR高/低表达的细胞株进行5-FU耐药性分析.结果显示,结肠癌病灶组织中pxr外显子3甲基化频率显著增加,伴有pxr,cyp3a4表达的增强.在结肠组织及结肠癌细胞系中,pxr与cyp3a4的表达均密切相关,且均与pxr甲基化程度相关.PXR高表达细胞株LS180对5-FU的耐药性显著升高,以siRNA分别下调pxr及cyp3a4的表达,均可增加LS180对5 -FU的敏感性.结果提示,pxr基因外显子3区甲基化与PXR及CYP3A4的高表达密切相关,并与结肠癌细胞对5-FU的抗药性相关.     

5-Fluorouracil Chemotherapy of Gastric Cancer Generates Residual Cells with Properties of Cancer Stem Cells

Background: 5-Fluorouracil (5Fu) chemotherapy is the first treatment of choice for advanced gastric cancer (GC), but its effectiveness is limited by drug resistance. Emerging evidence suggests that the existence of cancer stem cells (CSCs) contributes to chemoresistance. The aim of the present study was to determine whether 5Fu chemotherapy generates residual cells with CSC-like properties in GC. Methods: Human GC cell lines, SGC7901 and AGS, were exposed to increasing 5Fu concentrations. The residual cells were assessed for both chemosensitivity and CSC-like properties. B lymphoma Mo-MLV insertion region 1 (BMI1), a putative CSC protein, was analyzed by immunohistochemical staining and subjected to pairwise comparison in GC tissues treated with or without neoadjuvant 5Fu-based chemotherapy. The correlation between BMI1 expression and recurrence-free survival in GC patients who received 5Fu-based neoadjuvant chemotherapy was then examined. Results: The residual cells exhibited 5Fu chemoresistance. These 5Fu-resistant cells displayed some CSC features, such as a high percentage of quiescent cells, increased self-renewal ability and tumorigenicity. The 5Fu-resistant cells were also enriched with cells expressing cluster of differentiation (CD)133⁺, CD326⁺ and CD44⁺CD24^-. Moreover, the BMI1 gene was overexpressed in 5Fu-resistant cells, and BMI1 knockdown effectively reversed chemoresistance. The BMI1 protein was highly expressed consistently in the remaining GC tissues after 5Fu-based neoadjuvant chemotherapy, and BMI1 levels were correlated positively with recurrence-free survival in GC patients who received 5Fu-based neoadjuvant chemotherapy. Conclusions: Our data provided molecular evidence illustrating that 5Fu chemotherapy in GC resulted in acquisition of CSC-like properties. Moreover, enhanced BMI1 expression contributed to 5Fu resistance and may serve as a potential therapeutic target to reverse chemoresistance in GC patients.     

Elimination of Colon Cancer Stem-Like Cells by the Combination of Curcumin and FOLFOX

5-Fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) remains the backbone of colorectal cancer chemotherapeutics but with limited success. This could partly be due to the enrichment of cancer stem cells (CSCs) that are resistant to conventional chemotherapy. Therefore, validation of a nontoxic agent that can either cause reversal of chemoresistance or promote the killing of CSCs would be highly desirable. The current study examines whether curcumin, the major active ingredient of turmeric, either alone or together with FOLFOX, would be an effective strategy to eliminate colon CSCs. Exposure of colon cancer HCT-116 or HT-29 cells to FOLFOX that inhibited their growth led to the enrichment of CSC phenotype as evidenced by increased proportion of CD133-, CD44-, and/or CD166-positive cells and epidermal growth factor receptor (EGFR) levels. Treatment of FOLFOX-surviving colon cancer cells with either curcumin alone or together with FOLFOX resulted in a marked reduction in CSCs, as evidenced by the decreased expression of CD44 and CD166 as well as EGFR and by their ability to form anchorage-dependent colonies. They also caused disintegration of colonospheres. Increased expression of EGFR in FOLFOX-surviving cells could be attributed to hypomethylation of the EGFR promoter, whereas an opposite phenomenon was observed when the FOLFOX-surviving cells were treated with curcumin and/or FOLFOX. These changes were accompanied by parallel alterations in the levels of DNA methyltransferase 1. In conclusion, our data suggest that curcumin by itself or together with the conventional chemotherapeutic could be an effective treatment strategy for preventing the emergence of chemoresistant colon cancer cells by reducing/eliminating CSCs.     

Wnt-5b在结肠癌中的表达及对结肠癌细胞的影响

Wnt-5b是Wnt信号转导途径中一个重要的成员,其与Wnt-5a具有80%的同源性,目前国内对于其在肿瘤的表达与功能尚不十分清楚.通过免疫组化检测70例不同分化程度结肠癌和30例正常结肠组织中Wnt-5b的表达揭示,其在结肠癌组阳性率表达低于正常组,随肿瘤分化程度减低,有下降的趋势;蛋白质印迹法检测表明,Wnt-5b在高分化人结肠癌细胞株Caco-2表达量低于低分化人结肠癌细胞株RKO和正常人结肠细胞株NCM460;Wnt-5b可减低Caco-2中β-Catenin的表达,抑制细胞运动能力,增加细胞凋亡率.以上结果提示,Wnt-5b在结肠癌中很可能扮演抑制肿瘤的角色,与抑制β-Catenin的表达和肿瘤细胞的运动能力及促进细胞凋亡有关.     

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation¹. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity⁴. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types^5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.     

Curcumin Conjugated with PLGA Potentiates Sustainability,Anti-Proliferative Activity and Apoptosis in Human Colon Carcinoma Cells

Curcumin, an ingredient of turmeric, exhibits a variety of biological activities such as anti-inflammatory, anti-atherosclerotic, anti-proliferative, anti-oxidant, anti-cancer and anti-metastatic. It is a highly pleiotropic molecule that inhibits cell proliferation and induces apoptosis in cancer cells. Despite its imperative biological activities, chemical instability, photo-instability and poor bioavailability limits its utilization as an effective therapeutic agent. Therefore, enhancing the bioavailability of curcumin may improve its therapeutic index for clinical setting. In the present study, we have conjugated curcumin with a biodegradable polymer Poly (D, L-lactic-co-glycolic acid) and evaluated its apoptotic potential in human colon carcinoma cells (HCT 116). The results show that curcumin-PLGA conjugate efficiently inhibits cell proliferation and cell survival in human colon carcinoma cells as compared to native curcumin. Additionally, curcumin conjugated with PLGA shows improved cellular uptake and exhibits controlled release at physiological pH as compared to native curcumin. The curcumin-PLGA conjugate efficiently activates the cascade of caspases and promotes intrinsic apoptotic signaling. Thus, the results suggest that conjugation potentiates the sustainability, anti-proliferative and apoptotic activity of curcumin. This approach could be a promising strategy to improve the therapeutic index of cancer therapy.     

TRAF4调控人结直肠癌细胞增殖的机制研究

目的:研究TRAF4影响结直肠癌细胞增殖的分子机制。方法:MTS和软琼脂集落形成实验检测基因沉默TRAF4后对结直肠癌细胞生长及相关信号通路的影响,检测TRAF4表达下调后结直肠癌细胞的糖酵解变化及己糖激酶II的表达和结直肠细胞对5-Fu的敏感性。结果:基因沉默TRAF4抑制结直肠癌细胞的停泊依赖和停泊非依赖增殖,抑制EGF诱导的Akt活化,下调结直肠癌细胞糖酵解,抑制己糖激酶II的表达,增加结直肠癌细胞对5-Fu的敏感性。结论:TRAF4通过调控糖代谢影响结直肠癌细胞增殖。     

NDRG1对人肠癌细胞失巢凋亡的影响

目的:探讨NDRG1对体外培养的人肠癌细胞系失巣凋亡的影响。方法:采用慢病毒系统将NDRG1表达单元转入人肠癌细胞系SW620、HCT8中,建立相应的过表达稳定细胞系;通过siRNA的方法干扰HCT116和LOVO细胞系中NDRG1的表达,分别在非贴壁培养的情况下培养48小时,采用流式细胞术和TUNEL染色检测细胞的凋亡情况。结果:在贴壁培养条件下,NDRG1过表达并没有显著影响肠癌细胞的生长及增殖,而NDRG1特异性siRNA干扰HCT116细胞中NDRG1的表达后,其凋亡率无明显变化(P0.05)。在悬浮培养条件下,NDRG1过表达的肠癌细胞的失巢凋亡率显著低于正常对照组(P0.05),而用三种不同的siRNA干扰HCT116及LOVO细胞中NDRG1的表达后,其失巢凋亡率均显著高于正常对照组(P0.05)。结论:NDRG1在体外可抑制人肠癌细胞的失巢凋亡。     

Anti-Inflammatory Effect of Buckwheat Sprouts in Lipopolysaccharide-Activated Human Colon Cancer Cells and Mice

In conducting an in vitro screening of ethanol extracts from various natural foods using a human colon cancer cell line (CoLoTC cells), an extract of buckwheat sprouts (ExtBS) was found to express significant anti-inflammatory activity. The anti-inflammatory activity of ExtBS was confirmed by oral administration of lipopolysaccharide (LPS) to mice. Inflammatory cytokines (interleukin 6 and tumor necrosis factor alpha) were markedly up-regulated in the spleen and liver from LPS-administrated mice, and combinatory treatment with LPS and ExtBS decreased up-regulation of them in both cytokines. Both serum cytokine levels corresponded to their gene expressions in tissues, but no anti-inflammatry effect in mice was observed when ExtBS was treated intraperitoneally. ExtBS oral administration also showed protective activity as to hepatic injury induced by galactosamine/LPS treatment. Based on these data, we suggest that ExtBS contains anti-inflammatory compounds.     

抑制Hedgehog信号通路改变结肠癌细胞基因表达谱

Hedgehog（Hh）信号通路在机体发育和肿瘤发生中发挥着重要作用。在该研究中,Western blot检测三株结肠癌细胞Hedgehog信号通路组分的表达,结果表明三株结肠癌细胞中HT-29细胞Hedgehog信号通路组分较完整。采用MTT和BrdU法检测Hedgehog信号通路膜受体Smo特异性抑制剂环杷明和末端转录因子Gli1/2的特异性抑制剂GANT61对HT-29细胞的影响,提示这两种抑制剂均显著抑制HT-29细胞生存率和细胞增殖率,且GANT61比环杷明更敏感。表达谱芯片检测阻断Hedgehog信号通路后HT-29细胞基因谱的变化,结合生物信息学分析,揭示HT-29细胞经环杷明和GANT61处理后基因表达呈现抑制特征,其差异基因表达主要以下调为主,其中环杷明主要影响细胞内源刺激等,而GANT61主要影响代谢和类固醇合成,并与MAPK信号通路有关联,两者均能影响细胞免疫及凋亡相关通路。这些结果提示,Hh信号通路有可能作为结肠癌的治疗靶点。     

Apoptosis Induction by Wheat-flour Sphingoid Bases in DLD-1 Human Colon Cancer Cells

The apoptotic effects of plant sphingoid bases prepared from wheat-flour cerebroside on human colorectal cancer DLD-1 cells were examined. The viability of DLD-1 cells treated with such plant sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. Morphological changes such as condensed chromatin fragments were found, so those sphingoid bases reduced cell viability through causing apoptosis in these cells.     

Notch1 Is a 5-Fluorouracil Resistant and Poor Survival Marker in Human Esophagus Squamous Cell Carcinomas

Notch signaling involves the processes that govern cell proliferation, cell fate decision, cell differentiation and stem cell maintenance. Due to its fundamental role in stem cells, it has been speculated during the recent years that Notch family may have critical functions in cancer stem cells or cancer cells with a stem cell phenotype, therefore playing an important role in the process of oncogenesis. In this study, expression of Notch family in KYSE70, KYSE140 and KYSE450 squamous esophageal cancer cell lines and virus transformed squamous esophageal epithelial cell line Het-1A was examined by quantitative RT-PCR. Compared to the Het-1A cells, higher levels of Nocth1 and Notch3 expression in the cancer cell lines were identified. Due to the finding that NOTCH3 mainly mediates squamous cell differentiation, NOTCH1 expression was further studied in these cell lines. By Western blot analyses, the KYSE70 cell line which derived from a poorly differentiated tumor highly expressed Notch1, and the Notch1 expression in this cell line was hypoxia inducible, while the KYSE450 cell line which derived from a well differentiated tumor was always negative for Notch1, even in hypoxia. Additional studies demonstrated that the KYSE70 cell line was more 5-FU resistant than the KYSE450 cell line and such 5-FU resistance is correlated to Notch1 expression verified by Notch1 knockdown experiments. In clinical samples, Notch1 protein expression was detected in the basal cells of human esophagus epithelia, and its expression in squamous cell carcinomas was significantly associated with higher pathological grade and shorter overall survival. We conclude that Notch1 expression is associated with cell aggressiveness and 5-FU drug resistance in human esophageal squamous cell carcinoma cell lines in vitro and is significantly associated with a poor survival in human esophageal squamous cell carcinomas.     

巨噬细胞、内皮细胞对高密度脂蛋白的氧化修饰

为了探讨高密度脂蛋白 (HDL)在体内发生氧化修饰的部位及机制 ,分别观察了培养人动脉平滑肌细胞 (SMC)、动脉内皮细胞 (EC)及巨噬细胞 (MΦ)与HDL共同温育过程中 ,HDL的琼脂糖电泳相对迁移率 (REM)、硫代巴比妥酸反应物质 (TBARS)以及溶血卵磷脂 卵磷脂 (LPC PC)值等氧化指标的变化 .结果发现 ,HDL与 3种细胞温育 12h时 ,HDL几乎不发生氧化修饰 ,而HDL与MΦ和EC温育 2 4h后 ,其REM、TBARS、LPC PC值均显著上升 (p <0 0 1) ;而HDL与SMC温育后 ,其REM、TBARS值无显著改变 ,LPC PC值增加 (p <0 0 1) .结果提示 ,活体内HDL可能主要在动脉壁的内皮细胞及巨噬细胞的作用下发生氧化修饰     

Curcumin Modulates DNA Methylation in Colorectal Cancer Cells

Aim

Recent evidence suggests that several dietary polyphenols may exert their chemopreventive effect through epigenetic modifications. Curcumin is one of the most widely studied dietary chemopreventive agents for colon cancer prevention, however, its effects on epigenetic alterations, particularly DNA methylation, remain unclear. Using systematic genome-wide approaches, we aimed to elucidate the effect of curcumin on DNA methylation alterations in colorectal cancer cells.

Materials and Methods

To evaluate the effect of curcumin on DNA methylation, three CRC cell lines, HCT116, HT29 and RKO, were treated with curcumin. 5-aza-2′-deoxycytidine (5-aza-CdR) and trichostatin A treated cells were used as positive and negative controls for DNA methylation changes, respectively. Methylation status of LINE-1 repeat elements, DNA promoter methylation microarrays and gene expression arrays were used to assess global methylation and gene expression changes. Validation was performed using independent microarrays, quantitative bisulfite pyrosequencing, and qPCR.

Results

As expected, genome-wide methylation microarrays revealed significant DNA hypomethylation in 5-aza-CdR-treated cells (mean β-values of 0.12), however, non-significant changes in mean β-values were observed in curcumin-treated cells. In comparison to mock-treated cells, curcumin-induced DNA methylation alterations occurred in a time-dependent manner. In contrast to the generalized, non-specific global hypomethylation observed with 5-aza-CdR, curcumin treatment resulted in methylation changes at selected, partially-methylated loci, instead of fully-methylated CpG sites. DNA methylation alterations were supported by corresponding changes in gene expression at both up- and down-regulated genes in various CRC cell lines.

Conclusions

Our data provide previously unrecognized evidence for curcumin-mediated DNA methylation alterations as a potential mechanism of colon cancer chemoprevention. In contrast to non-specific global hypomethylation induced by 5-aza-CdR, curcumin-induced methylation changes occurred only in a subset of partially-methylated genes, which provides additional mechanistic insights into the potent chemopreventive effect of this dietary nutraceutical.     

Subverting ER-Stress towards Apoptosis by Nelfinavir and Curcumin Coexposure Augments Docetaxel Efficacy in Castration Resistant Prostate Cancer Cells

Despite its side-effects, docetaxel (DTX) remains a first-line treatment against castration resistant prostate cancer (CRPC). Therefore, strategies to increase its anti-tumor efficacy and decrease its side effects are critically needed. Targeting of the constitutive endoplasmic reticulum (ER) stress in cancer cells is being investigated as a chemosensitization approach. We hypothesized that the simultaneous induction of ER-stress and suppression of PI3K/AKT survival pathway will be a more effective approach. In a CRPC cell line, C4-2B, we observed significant (p<0.005) enhancement of DTX-induced cytotoxicity following coexposure to thapsigargin and an AKT-inhibitor. However, since these two agents are not clinically approved, we investigated whether a combination of nelfinavir (NFR) and curcumin (CUR), known to target both these metabolic pathways, can similarly increase DTX cytotoxicity in CRPC cells. Within 24 hrs post-exposure to physiologic concentrations of NFR (5 µM) and CUR (5 µM) a significantly (p<0.005) enhanced cytotoxicity was evident with low concentration of DTX (10 nM). This 3-drug combination rapidly increased apoptosis in aggressive C4-2B cells, but not in RWPE-1 cells or in primary prostate epithelial cells (PrEC). Comparative molecular studies revealed that this 3-drug combination caused a more pronounced suppression of phosphorylated-AKT and higher induction in phosphorylated-eIF2α in C4-2B cells, as compared to RWPE-1 cells. Acute exposure (3–9 hrs) to this 3-drug combination intensified ER-stress induced pro-apoptotic markers, i.e. ATF4, CHOP, and TRIB3. At much lower concentrations, chronic (3 wks) exposures to these three agents drastically reduced colony forming units (CFU) by C4-2B cells. In vivo studies using mice containing C4-2B tumor xenografts showed significant (p<0.05) enhancement of DTX’s (10 mg/kg) anti-tumor efficacy following coexposure to NFR (20 mg/kg) & CUR (100 mg/kg). Immunohistochemical (IHC) analyses of tumor sections indicated decreased Ki-67 staining and increased TUNEL intensity in mice exposed to the 3-drug combination. Therefore, subverting ER-stress towards apoptosis using adjuvant therapy with NFR and CUR can chemosensitize the CRPC cells to DTX therapy.