Chemosensitivity Of Pancreatic Cancer Cells Research Articles

Heptadecanoic Acid, an Odd-Chain Fatty Acid, Induces Apoptosis and Enhances Gemcitabine Chemosensitivity in Pancreatic Cancer Cells.

Odd-chain saturated fatty acids generally serve as specific biomarkers of dietary components and dairy intake, some of which have anticancer properties. This study was performed to assess the anticancer effects of heptadecanoic acid (HDNA) in human pancreatic carcinoma cells. MTT (thiazolyl blue tetrazolium bromide) assay showed that HDNA exerted stronger cytotoxic effects than pentadecanoic acid, palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), and linoleic acid (18:2) on both Panc-1 and MIA PaCa-2 pancreatic cancer cells. In addition, HDNA reduced colony formation and induced apoptosis in these pancreatic cancer cells as indicated by Hoechst 33342 staining, Annexin V/propidium iodide staining, cell cycle analysis, and Western blotting analysis in a dose-dependent manner. Moreover, HDNA synergistically reduced cell viability and promoted apoptosis when combined with gemcitabine (GEM), a chemotherapeutic agent commonly used in the treatment of pancreatic cancer. GEM-resistant MIA PaCa-2 (GR-MIA PaCa-2) cells with a resistance indices (RI) value of 215.09 [RI = half-maximal inhibitory concentration (IC50) of GR-MIA PaCa-2 cells/IC50 of MIA PaCa-2 cells] were established, and the efficacy of HDNA on GEM chemosensitivity was confirmed. Surprisingly, HDNA exhibited even higher antiproliferative efficacy against GR-MIA PaCa-2 cells (IC50 = 71.45 ± 6.37 μM) than parental MIA PaCa-2 cells (IC50 = 77.47 ± 2.10 μM). Finally, HDNA treatment inhibited the Hippo pathway and induced apoptosis of GR-MIA PaCa-2 cells. These findings suggest the beneficial effects of a HDNA-rich diet during pancreatic cancer treatments.

Predicting the chemosensitivity of pancreatic cancer cells as a personalized therapy.

Annually, approx. 4000 patients are diagnosed with pancreatic cancer in Poland, and the number of deaths is close to the number of diagnoses. Such a high morbidity/mortality ratio is caused by a high percentage of unresectable lesions (about 80%) and chemoresistance, which, among other things, is due to the specific desmoplastic environment. Currently, there are 2 main systemic treatment regimens for pancreatic cancer: FOLFIRINOX (which is a combination of folic acid, fluorouracil (5-FU), irinotecan, and oxaliplatin) and combined treatment with nab-paclitaxel plus gemcitabine (NPXL+GMC). In order to increase the effectiveness of systemic treatments for individual patients, cell lines derived from resected pancreatic tumors were developed and their chemosensitivity to various agents was examined. The hypothesis was that patients may benefit from individualization of chemotherapy. Patients with histopathologically confirmed pancreatic cancer were operated on using irreversible electroporation (IRE) procedure. After isolating and establishing individual cell lines, chemosensitivity to 5-FU, GMC and NPXL was determined using MTT assay in primary and metastatic cell cultures. Three primary cell lines were isolated for the prediction of chemosensitivity. Gemcitabine was shown to be more effective at lower doses compared to 5-FU, while NPXL was more effective than 5-FU, and both of these were less effective in metastatic cells. Pancreatic cancer cell chemoresistance was confirmed in stage IV. Determination of chemosensitivity profiles using cell lines may help in the selection of systemic treatments for individual patients. This method can be the basis for a personalized planned chemotherapeutic protocol.

Overexpression of DUOX2 mediates doxorubicin resistance and predicts prognosis of pancreatic cancer.

Dysregulation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) family is frequently observed in cancers and associated with their development and progression. However, the expression, role, and clinical significance of the NOX family members in pancreatic cancer remain unexplored. The expression levels of the 7 NOX family genes were analyzed in Gene Expression Omnibus (GEO) datasets. The messenger RNA (mRNA) expression and gene alterations were explored using The Cancer Genome Atlas (TCGA) data portal. Clinical significance analyses of the NOX family genes were conducted among pancreatic cancer patients. The expression and prognostic value of dual oxidase 2 (DUOX2) were then validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) in an independent validation cohort. The function of DUOX2 was analyzed by gene set enrichment analysis (GSEA) and its effect on the chemosensitivity of pancreatic cancer cells was detected by Cell Counting Kit-8 (CCK-8) assay. Results showed that NOX1, NOX2 (CYBB), NOX4, DUOX1, and DUOX2 were upregulated, while NOX3 and NOX5 were downregulated in pancreatic cancer tissues compared with nontumor tissues. Genomic alteration analysis demonstrated that deregulation of NOX family genes was partially caused by genomic alterations. Survival analyses showed that only DUOX2 was associated with overall survival (OS) and relapse-free survival (RFS) of pancreatic cancer patients. The DUOX2 gene was observed to be markedly overexpressed in pancreatic cancer. In the GSEA results for pancreatic cancer patients, DUOX2 was significantly associated with oxidoreductase activity acting on nicotinamide adenine dinucleotide hydrogen (NADH) or NADPH and uridine 5'-diphospho-glucuronosyltansferase (UDP) glycosyltransferase activity. Knockdown of DUOX2 in pancreatic cancer cells increased their sensitivity to doxorubicin. Overexpression of DUOX2 is correlated with prognosis and recurrence in pancreatic cancer patients and acts as a good marker for pancreatic cancer course prediction; furthermore, DUOX2 might be a therapeutic target for pancreatic cancer patients.

Panax notoginseng Saponins Promote Cell Death and Chemosensitivity in Pancreatic Cancer through the Apoptosis and Autophagy Pathways.

Panax notoginseng Saponins (PNS) is used as a traditional Chinese medicine for ischemic stroke and cardiovascular disease; it has been proven to possess anticancer activity recently. In this study, we aimed to explore the curative anticancer effect and potential mechanisms of PNS in pancreatic cancer cells. Pancreatic cancer Miapaca2 and PANC-1 cells were treated with PNS and Gemcitabine (Gem), respectively. Then the cell viability was assessed by CCK-8 assay, cell proliferation was tested by colony formation assay and EdU cell proliferation assay, cell migration and invasiveness were tested by wound healing assay and transwell assay, respectively, and cell apoptosis was detected by flow cytometry. Finally, we detected the expression levels of proteins related to migration, apoptosis and autophagy through Western blotting. PNS not only inhibited the proliferation, migration, invasion and autophagy of Miapaca2 and PANC-1 cells, but also induced apoptosis and promoted chemosensitivity of pancreatic cancer cells to Gem. PNS may exhibit cytotoxicity and increase chemosensitivity of pancreatic cancer cells to Gem by inhibiting autophagy and inducing apoptosis, providing a new strategy and potential treatment option for pancreatic cancer.

N-acetyl cysteine induces quiescent-like pancreatic stellate cells from an active state and attenuates cancer-stroma interactions

BackgroundPancreatic stellate cells (PSCs) occupy the majority of the pancreatic cancer microenvironment, contributing to aggressive behavior of pancreatic cancer cells (PCCs). Recently, anti-fibrotic agents have proven to be an effective strategy against cancer, but clinical trials have shown little efficacy, and the driving mechanism remains unknown. N-acetyl-cysteine (NAC) is often used for pulmonary cystic fibrosis. Pioglitazone, an agonist of peroxisome proliferator-activated receptor gamma, was habitually used for type II diabetes, but recently reported to inhibit metastasis of PCCs. However, few studies have focused on the effects of these two agents on cancer-stromal interactions.MethodWe evaluated the expression of α-smooth muscle actin (α-SMA) and the number of lipid droplets in PSCs cultured with or without NAC. We also evaluated changes in invasiveness, viability, and oxidative level in PSCs and PCCs after NAC treatment. Using an indirect co-culture system, we investigated changes in viability, invasiveness, and migration of PSCs and PCCs. Combined treatment effects of NAC and Pioglitazone were evaluated in PSCs and PCCs. In vivo, we co-transplanted KPC-derived organoids and PSCs to evaluate the effects of NAC and Pioglitazone’s combination therapy on subcutaneous tumor formation and splenic xenografted mouse models.ResultsIn vitro, NAC inhibited the viability, invasiveness, and migration of PSCs at a low concentration, but not those of PCCs. NAC treatment significantly reduced oxidative stress level and expression of α-SMA, collagen type I in PSCs, which apparently present a quiescent-like state with a high number of lipid droplets. Co-cultured PSCs and PCCs mutually promoted the viability, invasiveness, and migration of each other. However, these promotion effects were attenuated by NAC treatment. Pioglitazone maintained the NAC-induced quiescent-like state of PSCs, which were reactivated by PCC-supernatant, and enhanced chemosensitivity of PCCs. In vivo, NAC and Pioglitazone’s combination suppressed tumor growth and liver metastasis with fewer stromal components and oxidative stress level.ConclusionNAC suppressed activated PSCs and attenuated cancer-stromal interactions. NAC induces quiescent-like PSCs that were maintained in this state by pioglitazone treatment.

Metformin Increases the Chemosensitivity of Pancreatic Cancer Cells to Gemcitabine by Reversing EMT Through Regulation DNA Methylation of miR-663.

BackgroundPancreatic cancer is a devastating malignancy with poor prognosis. Metformin, a classic anti-diabetes drug, seems to improve survival of pancreatic cancer patients in some studies.MethodsCell counting kit-8 assay was used to detect the BxPC-3 and MIAPaCa-2 cell viability after treatment with gemcitabine only or with different concentrations of metformin. The methylation state and expression level of miR-663 were detected by methylation analysis and RT-PCR. Dual-luciferase reporter gene analysis, Western blot and RT-PCR were used to confirm the target of miR-663. Moreover, xenograft experiment was also performed to validate the role of metformin in chemosensitivity in vivo.ResultsWe found that metformin increased the chemosensitivity of pancreatic cancer cells to gemcitabine, and epithelial–mesenchymal transition (EMT) progress caused by gemcitabine was suppressed by metformin. We further explored the possible molecular mechanisms and it was demonstrated that CpG islands of miR-663 were hypomethylated and relative expression level of miR-663 was up-regulated after treatment of metformin. miR-663, an important cancer suppressor miRNA, was confirmed to increase the chemosensitivity of pancreatic cancer cells by reversing EMT directly targeted TGF-β1. Moreover, we identified that metformin increased the chemosensitivity through up-regulating expression of miR-663.ConclusionWe demonstrated that metformin increased the chemosensitivity of pancreatic cancer cells to gemcitabine by reversing EMT through regulation DNA methylation of miR-663.

Ursolic acid promotes apoptosis, autophagy, and chemosensitivity in gemcitabine-resistant human pancreatic cancer cells.

Gemcitabine (GEM) resistance in pancreatic adenocarcinoma mediated by the receptor for advanced glycation end products (RAGE) has been demonstrated. Therefore, investigating the safety and the potential of new auxiliary methods for pancreatic cancer treatment is urgent. Ursolic acid (UA), a natural pentacyclic triterpenoid found in apple peels, rosemary, and thyme, has been reported to have anticancer capacity. This study aimed to reveal the underlying mechanisms of UA in cell death and drug enhancement, especially in GEM-resistant pancreatic cancer cells. First, GEM-resistant cells (MIA Paca-2GEMR cells) were established by incrementally increasing GEM culture concentrations. UA treatment reduced cell viability through cell cycle arrest and endoplasmic reticulum (ER) stress, resulting in apoptosis and autophagy in a dose-dependent manner in MIA Paca-2 and MIA Paca-2GEMR cells. High RAGE expression in MIA Paca-2GEMR cells was suppressed by UA treatment. Interestingly, knocking down RAGE expression showed similar UA-induced effects in both cell lines. Remarkably, UA had a drug-enhancing effect by decreasing cell viability and increasing cell cytotoxicity when combined with GEM treatment. In conclusions, UA triggered ER stress, subsequently regulating apoptosis- and autophagy-related pathways and increasing GEM chemosensitivity in pancreatic cancer cells by inhibiting the expression of RAGE.

Activation of GLP-1 receptor enhances the chemosensitivity of pancreatic cancer cells.

This study aimed to determine whether and how the glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide affects the chemoresistance and chemosensitivity of pancreatic cancer cells to gemcitabine in vitro and in vivo. The GLP-1R and protein kinase A (PKA) levels were compared between the human pancreatic cancer cell line PANC-1 and the gemcitabine-resistant cell line PANC-GR. The in vitro effects of liraglutide on the cell proliferation and apoptosis as well as the nuclear factor-kappa B NF-κB expression levels of PANC-GR cells were evaluated. In addition, a mouse xenograft model of human pancreatic cancer was established by s.c. injection of PANC-1 cells, and the effects of liraglutide on the chemosensitivity were evaluated in vitro and in vivo. In contrast to PANC-1 cells, PANC-GR cells exhibited lower expression levels of GLP-1R and PKA. Incubation with liraglutide dose dependently inhibited the growth, promoted the apoptosis, and increased the expression of GLP-1R and PKA of PANC-GR cells. Similar effects of liraglutide were observed in another human pancreatic cancer cell line MiaPaCa-2/MiaPaCa-2-GR. Either the GLP-1R antagonist Ex-9, the PKA inhibitor H89, or the NF-κB activator lipopolysaccharide (LPS) could abolish the antiproliferative and proapoptotic activities of liraglutide. Additionally, each of these agents could reverse the expression of NF-κB and ABCG2, which was decreased by liraglutide treatment. Furthermore, liraglutide treatment increased the chemosensitivity of pancreatic cancer cells to gemcitabine, as evidenced by in vitro and in vivo experiments. Thus, GLP-1R agonists are safe and beneficial for patients complicated with pancreatic cancer and diabetes, especially for gemcitabine-resistant pancreatic cancer.

LncRNA H19-Derived miR-675-3p Promotes Epithelial-Mesenchymal Transition and Stemness in Human Pancreatic Cancer Cells by targeting the STAT3 Pathway.

Objective: The functional role and mechanism of the long noncoding RNA (lncRNA) H19 in regulating human pancreatic cancer (PC) cell stemness and invasion have not been completely elucidated. This study aimed to evaluate the role of H19 in regulating the stemness, epithelial-mesenchymal transition (EMT), invasion and chemosensitivity of PC cells.Methods: The sphere-forming ability was assessed using serum-free floating-culture systems. Chemosensitivity was evaluated via CCK-8 and flow cytometry assays in vitro. Migration and invasion were evaluated by transwell assays. The expression of stemness and EMT markers was detected by flow cytometry, qRT-PCR and western blot analyses. Xenograft initiation, growth and sensitivity were examined; Ki-67 nuclear staining intensity was evaluated by immunohistochemistry; and in situ apoptosis was evaluated by a TUNEL assay.Results: H19 played an important role in maintaining PC cell stemness. Upregulated H19 expression in CAPAN-1 cells promoted tumor cell migration, invasion, EMT and chemoresistance. In contrast, downregulated H19 expression in PANC-1 cells yielded the opposite results. These effects were mediated by positively modulating the STAT3 pathway. Furthermore, SOCS5, an endogenous inhibitor of the STAT3 pathway, was a direct target of miR-675-3p, which was positively regulated by H19 in PC cells.Conclusions: The H19/miR-675-3p signaling axis plays a critical role in maintaining the EMT process and stemness of PC cells by directly targeting SOCS5 to activate the STAT3 pathway. These data provide new insights into the oncogenic function of H19 in human PC and reveal potential targets for the development of optimal treatment approaches for this disease.

Quercetin facilitates cell death and chemosensitivity through RAGE/PI3K/AKT/mTOR axis in human pancreatic cancer cells

The triggering of gemcitabine (GEM) drug resistance in pancreatic cancer by the receptor for advanced glycation end products (RAGE) has been demonstrated. Hence, finding a safe and effective adjuvant for preventing pancreatic cancer progression is imperative. Quercetin is a flavonoid that is abundant in apples, grapes, red raspberry, and onions and has been reported to inhibit RAGE. This research aimed to investigate the mechanisms of quercetin in regulating cell death and enhancing drug effects through RAGE reduction, especially in GEM-resistant pancreatic cancer cells. Our results showed that silencing RAGE expression by RAGE-specific siRNA transfection significantly increased cell death by apoptosis, autophagy and GEM-induced cytotoxicity by suppressing the PI3K/AKT/mTOR axis in MIA Paca-2 and MIA Paca-2 GEMR cells (GEM-resistant cells). Notably, quercetin showed a dramatic effect similar to RAGE silencing that effectively attenuated RAGE expression to facilitate cell cycle arrest, autophagy, apoptosis, and GEM chemosensitivity in MIA Paca-2 GEMR cells, suggesting that an additional reaction occurred under combined quercetin and GEM treatment. In conclusion, the results demonstrated that the molecular mechanisms of quercetin in regulating apoptosis and autophagy-related pathways and increasing GEM chemosensitivity in pancreatic cancer cells involved inhibition of RAGE expression.

Alantolactone induces apoptosis and improves chemosensitivity of pancreatic cancer cells by impairment of autophagy-lysosome pathway via targeting TFEB

The lysosome is emerging as a central regulator of the autophagic process, which plays a critical role in tumor growth and chemoresistance. Alantolactone, which is a natural compound produced by Inula helenium, has been shown to induce apoptosis in numerous cancer types. However, the mechanism by which alantolactone regulates apoptosis is still poorly understood. In this work, we observed that alantolactone caused the accumulation of autophagosomes due to impaired autophagic degradation and substantially inhibited the activity and expression of CTSB/CTSD proteins that when depleted caused lysosomal dysfunction. Furthermore, we found that alantolactone inhibited the proliferation of pancreatic cancer cells in vitro and in vivo and enhanced the chemosensitivity of pancreatic cancer cells to oxaliplatin. In addition, a reduction in TFEB levels was a critical event in the apoptosis and cell death caused by alantolactone. Our data demonstrated that alantolactone, which impaired autophagic degradation, was a pharmacological inhibitor of autophagy in pancreatic cancer cells and markedly enhanced the chemosensitivity of pancreatic cancer cells to oxaliplatin.

Demethylzeylasteral (ZST93) inhibits cell growth and enhances cell chemosensitivity to gemcitabine in human pancreatic cancer cells via apoptotic and autophagic pathways

The overall 5-year survival rate of patients with human pancreatic cancer remains less than 8% because of its aggressive growth, early metastasis and resistance to conventional chemoradiotherapy. It is essential to develop innovative and effective therapeutic agents to improve its prognosis. Demethylzeylasteral (ZST93) is a novel triterpenoid monomer extracted from the xylem of Tripterygium roots. Our study aimed to assess the effects of ZST93 on cell proliferation and its role in the chemosensitivity to gemcitabine in human pancreatic cancer cells. The effects of ZST93 on cancer cell proliferation, cell cycle distribution, apoptosis and autophagy were evaluated in various human pancreatic cancer cell lines, and the antitumor effects of ZST93 alone and in combination with gemcitabine were identified in a xenograft mouse model. The results showed that ZST93 could inhibit the proliferation of pancreatic cancer cells and arrest cell cycle at G0/G1 phase by regulating the expression of Cyclin D1 and Cyclin A2. Moreover, ZST93 killed pancreatic cancer cells through two different mechanisms: inducing autophagic cell death at low concentrations and apoptotic cell death at high concentrations. Furthermore, ZST93 could enhance the chemosensitivity of pancreatic cancer cells to gemcitabine both in vitro and in vivo through modulation of the cross talk between autophagy and apoptosis. ZST93 is a potential therapeutic agent for developing novel therapeutic strategies in human pancreatic cancer.

MicroRNA-29c Increases the Chemosensitivity of Pancreatic Cancer Cells by Inhibiting USP22 Mediated Autophagy

Background/Aims: Pancreatic cancer (PC) is an aggressive malignancy with a poor survival rate. Despite advances in the treatment of PC, the efficacy of therapy is limited by the development of chemoresistance. Here, we examined the role of microRNA-29c (miR-29c) and the involvement of autophagy and apoptosis in the chemoresistance of PC cells in vivo and in vitro. Methods: We employed qRT-PCR, western blot and immunofluorescence to examine the expression level of miR-29c, USP22 and autophagy relative protein. In addition, we used MTT assay to detect cell proliferation and transwell assay to measure migration and invasiveness. The apoptosis was determined using annexin V-FITC/PI apoptosis detection kit by flow cytometry. Luciferase reporter assays confirmed the relationship between USP22 and miR-29c. Results: miR-29c overexpression in the PC cell line PANC-1 enhanced the effect of gemcitabine on decreasing cell viability and inducing apoptosis and inhibited autophagy, as shown by western blotting, immunofluorescence staining, colony formation assays, and flow cytometry. Ubiquitin specific peptidase (USP)-22, a deubiquitinating enzyme known to induce autophagy and promote PC cell survival, was identified as a direct target of miR-29c. USP22 knockdown experiments indicated that USP22 suppresses gemcitabine-induced apoptosis by promoting autophagy, thereby increasing the chemoresistance of PC cells. Luciferase reporter assays confirmed that USP22 is a direct target of miR-29c. A xenograft mouse model demonstrated that miR-29c increases the chemosensitivity of PC in vivo by downregulating USP22, leading to the inhibition of autophagy and induction of apoptosis. Conclusions: Taken together, these findings reveal a potential mechanism underlying the chemoresistance of PC cells mediated by the regulation of USP22-mediated autophagy by miR-29c, suggesting potential targets and therapeutic strategies in PC.

Biodegradable nanocarriers for small interfering ribonucleic acid (siRNA) co-delivery strategy increase the chemosensitivity of pancreatic cancer cells to gemcitabine

Ribonucleic acid (RNA) interference (RNAi) therapies are promising cancer treatment modalities that can specifically target abnormal proto-oncogenes, thus improving the therapeutic effect. For the treatment of pancreatic cancer, targeting one mutant proto-oncogene by RNAi usually does not yield the desired therapeutic efficiency. Both K-ras gene mutations and Notch1 overexpression are common symptoms in pancreatic cancer patients, and play a crucial role in pancreatic cancer cell drug resistance. In this study, biodegradable charged polyester-based vectors (BCPVs) were synthesized for the co-delivery of K-ras and Notch1 small interfering ribonucleic acid (siRNA) into MiaPaCa-2 cells (pancreatic cancer cell line) to overcome drug resistance to gemcitabine (GEM), a first-line chemotherapeutic drug used in the clinic. BCPVs could effectively absorb negative siRNA to form a capsule-like structure, prevent siRNA from nuclease digestion in the serum, and promote effective siRNA cell internalization and endosomal escape. Through K-ras and Notch1 gene silencing in MiaPaCa-2 cells, BCPV-siRNAK-ras-siRNANotch1 nanocomplexes effectively reversed the epithelia-mesenchymal transition (EMT) in MiaPaCa-2 cells, thereby greatly enhancing the sensitivity of MiaPaCa-2 cells to GEM. MiaPaCa-2 cell proliferation, migration, and invasion were effectively inhibited, and cell apoptosis was also significantly enhanced by the synergistic antitumor effect of BCPV-siRNAK-ras-siRNANotch1 nanocomplexes and GEM. These results suggest that this combination RNAi therapy can be used to improve cancer cell sensitivity to chemotherapeutic drugs. Specifically, this newly developed strategy has a great potential for treating pancreatic cancer.

FBW7 increases the chemosensitivity of pancreatic cancer cells to gemcitabine through upregulation of ENT1.

F-box and WD repeat domain-containing 7 (FBW7) has been characterized as a tumor suppressor, and its mutation or decreased expression has been observed in many types of human cancers. Our recent studies have uncovered that in pancreatic cancer, the KRAS mutation decreased FBW7 expression through phosphorylation and subsequent ubiquitination. Moreover, FBW7 inhibited aerobic glycolysis in pancreatic cancer via induction of thioredoxin-interacting protein (TXNIP), a mitochondrial localized tumor suppressor. The roles of FBW7 in anti-apoptosis and drug resistance via proteosomal degradation of myeloid cell leukemia-1 (MCL-1), which is an anti-apoptotic factor have been reported. However, the role of FBW7 in the chemotherapeutic resistance of pancreatic cancer to gemcitabine has seldom been reported. In the present study, we demonstrated that overexpression of FBW7 in pancreatic cancer cells rendered increased sensitivity to gemcitabine. Mechanistically, FBW7 promoted gemcitabine sensitivity via upregulation of equilibrative nucleoside transporter 1 (ENT1) at the protein level rather than the transcriptional level. In depth analysis demonstrated that the ENT1 protein level could be increased by lysosome inhibition. Taken together, our results demonstrated that FBW7 could be a target for improving the therapeutic efficacy of gemcitabine by induction of ENT1.

Delineating the Role of βIV-Tubulins in Pancreatic Cancer: βIVb-Tubulin Inhibition Sensitizes Pancreatic Cancer Cells to Vinca Alkaloids

Pancreatic cancer (PC) is a lethal disease which is characterized by chemoresistance. Components of the cell cytoskeleton are therapeutic targets in cancer. βIV-tubulin is one such component that has two isotypes—βIVa and βIVb. βIVa and βIVb isotypes only differ in two amino acids at their C-terminus. Studies have implicated βIVa-tubulin or βIVb-tubulin expression with chemoresistance in prostate, breast, ovarian and lung cancer. However, no studies have examined the role of βIV-tubulin in PC or attempted to identify isotype specific roles in regulating cancer cell growth and chemosensitivity. We aimed to determine the role of βIVa- or βIVb-tubulin on PC growth and chemosensitivity. PC cells (MiaPaCa-2, HPAF-II, AsPC1) were treated with siRNA (control, βIVa-tubulin or βIVb-tubulin). The ability of PC cells to form colonies in the presence or absence of chemotherapy was measured by clonogenic assays. Inhibition of βIVa-tubulin in PC cells had no effect chemosensitivity. In contrast, inhibition of βIVb-tubulin in PC cells sensitized to vinca alkaloids (Vincristine, Vinorelbine and Vinblastine), which was accompanied by increased apoptosis and enhanced cell cycle arrest. We show for the first time that βIVb-tubulin, but not βIVa-tubulin, plays a role in regulating vinca alkaloid chemosensitivity in PC cells. The results from this study suggest βIVb-tubulin may be a novel therapeutic target and predictor of vinca alkaloid sensitivity for PC and warrants further investigation.

Downregulated miR-506 expression facilitates pancreatic cancer progression and chemoresistance via SPHK1/Akt/NF-κB signaling.

The aberrant expression of microRNAs (miRNAs) has emerged as an important hallmark of cancer. However, the molecular mechanisms underlying the changes in miRNA expression remain unclear. In this study, we discovered a novel epigenetic mechanism of miR-506 regulation and investigated its functional significance in pancreatic cancer. Sequencing analysis revealed that the miR-506 promoter is highly methylated in pancreatic cancer tissues compared with non-cancerous tissues. Reduced miR-506 expression was significantly associated with clinical stage, pathologic tumor status, distant metastasis and decreased survival of pancreatic cancer patients. miR-506 inhibited cell proliferation, induced cell cycle arrest at the G1/S transition and enhanced apoptosis and chemosensitivity of pancreatic cancer cells. Furthermore, we identified sphingosine kinase 1 (SPHK1) as a novel target of miR-506, the expression of which inhibited the SPHK1/Akt/NF-κB signaling pathway, which is activated in pancreatic cancer. High SPHK1 expression was significantly associated with poor survival in a large cohort of pancreatic cancer specimens. Our data suggest that miR-506 acts as a tumor suppressor miRNA and is epigenetically silenced in pancreatic cancer. The newly identified miR-506/SPHK1 axis represents a novel therapeutic strategy for future pancreatic cancer treatment.

Secreted protein acidic and rich in cysteine enhances the chemosensitivity of pancreatic cancer cells to gemcitabine

It has been previously shown that the simultaneous exposure of colon cancer cells MIP to irinotecan and secreted protein acidic and rich in cysteine (SPARC) enhances anticancer activity. However, whether there is same effect of SPARC in pancreatic cancer remains largely unknown. Therefore in this study, we aimed to investigate the role of SPARC played in the sensitivity of pancreatic cancer to gemcitabine. We first treated MIAPaCa2 and MIAPaCa2/SPARC69 cells with different concentrations of gemcitabine (2, 5, 10, and 20μM) for 24, 48, and 72h and selected the appropriated concentration for further study. Then we analyzed cell viability, cell cycle, and apoptosis and the levels of apoptosis-related proteins by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, fluorescence-activated cell sorting and Western blot were used, respectively. In this study, we found that gemcitabine inhibited the proliferation of pancreatic cancer cells in a time- and dose-dependent manner. Overexpression of SPARC increased the inhibiting effect of gemcitabine on pancreatic cancer cells. The colony size of MIAPaCa2/SPARC69 was much smaller than that of MIAPaCa2/V. There was a G0/G1 arrest with significant increase of apoptosis after gemcitabine treatment in MIAPaCa2/SPARC69 cells. Furthermore, our results demonstrated that overexpression of SPARC markedly increased the levels of pro-apoptotic proteins in gemcitabine-treated pancreatic cancer cells. The SPARC can enhance the chemosensitivity of pancreatic cancer cells to gemcitabine via regulating the expression of apoptosis-related proteins. These results have shown that the SPARC/ gemcitabine combination treatment may be a potentially useful therapeutic option for individuals diagnosed with pancreatic cancer.

Mechanism of SPARC-enhanced chemosensitivity of pancreatic cancer cells to gemcitabine

The aim of this study was to explore the effect of SPARC on the anti-cancer effect of gemcitabine and underlying mechanism in pancreatic cancer. After treating with gemcitabine, the proliferation rate of MIA PaCa2, MIA PaCa2/V and MIA PaCa2/SPARC69 cells was detected by MTT assay. The cell cycle distribution and cell apoptosis in each group were examined by flow cytometry, and the capability of clone formation was tested by adhesion-dependent clone formation assay. The apoptosis-related proteins were analyzed by Western blot. The growth of pancreatic cancer cells was inhibited by gemcitabine in a time-dependent and dose-dependent manner. Its IC50 at 24, 48, and 72-h was (40.1 ± 2.5) µmol/L, (15.0 ± 0.5) µmol/L and (6.6 ± 0.1) µmol/L, respectively. The overexpression of SPARC increased the inhibitory effect of gemcitabine on growth of pancreatic cancer MIA PaCa2/SPARC69 cells, presenting a dose- and time- dependent manner. Its IC50 at 24, 48, 72 h was (24.3 ± 1.5) µmol/L, (7.7 ± 0.3) µmol/L and (4.8 ± 0.2) µmol/L, respectively. The clone formation assay showed that before gemcitabine treatment, the clone numbers of MIA PaCa2, MIA PaCa2/V and MIA PaCa2/SPARC69 cells were (2350 ± 125), (2130 ± 120) and (1567 ± 11), respectively. After gemcitabine treatment, the clone numbers of MIA PaCa2, MIA PaCa2/V and MIA PaCa2/SPARC69 cells were ( 1674 ± 79) , (1587 ± 94) and (557 ± 61), respectively. The overexpression of SPARC enhanced the chemosensitivity of MIA PaCa2 cells to gemcitabine chemotherapy. After treating with 10 µmol/L gemcitabine for 48 h, the ratio of G0/G1 cells in MIA PaCa2, MIA PaCa2/V and MIA PaCa2/SPARC69 cells were (56.0 ± 5.5)%, (55.0 ± 4.5)% and (68.0 ± 7.0)%, respectively. The cells arrested at G0/G1 phase were significantly increased in the MIA PaCa2/SPARC69 cells. The apoptosis rates of MIA PaCa2, MIA PaCa2/V and MIA PaCa2/SPARC69 cells were (22.4 ± 2.5)%, (19.9 ± 2.0)% and (37.7 ± 3.9)%, respectively, indicating that overexpression of SPARC enhanced the gemcitabine-induced apoptosis in MIA PaCa2 cells. The Western blot analysis showed that, compared with MIA PaCa2 and MIA PaCa2/V cells, the expression of caspase-2, -8, -9 and cleaved PARP protein was significantly increased, while the expression of Bcl-2 was not changed significantly in the MIA PaCa2/SPARC69 cells. SPARC can enhance the chemosensitivity of pancreatic cancer cells to gemcitabine via regulating the expression of apoptosis-related proteins.

Inhibition of Cell Proliferation and Increase of Chemosensitivity by Simultaneous Knockdown of XIAP and Survivin in Pancreatic Carcinoma Cells

At present, classic therapies provide limited benefits to the survival of patients with pancreatic cancer. However, clinically available gene therapy strategies have not been well established. This study investigates the effect of shRNA-mediated inhibition of XIAP and survivin expression on the proliferation, apoptosis, and chemosensitivity of pancreatic cancer cells. Stable inhibition of XIAP and survivin expression in SW1990 and Panc-1 pancreatic cancer cells was established by lentivirus-carried shRNAs. The mRNA and protein expression of XIAP and survivin were detected by real-time PCR and Western blot, respectively. Cell proliferation was measured by MTT assay, and apoptosis was detected by caspase-3/7 activity and Hoechst33342 staining. The lentivirus-carried shRNA significantly inhibited XIAP and survivin expression. Simultaneous inhibition of XIAP and survivin expression in pancreatic cells significantly reduced cell proliferation, increased caspase-3/7 activity, and increased cell sensitization to 5-FU and gemcitabine treatments compared to inhibition of XIAP or survivin expression alone. However, simultaneous silencing of XIAP and survivin showed no significant difference in inducing cell apoptosis compared to silencing of XIAP or survivin expression alone. Simultaneous inhibition of XIAP and survivin expression may be an effective strategy for gene therapy of pancreatic cancer.