Histone deacetylase inhibitor augments anti-tumor effect of gemcitabine and pegylated interferon-α on pancreatic cancer cells

Background  

Histone deacetylase (HDAC) is strongly associated with epigenetic regulation and carcinogenesis, and its inhibitor can induce the differentiation or apoptosis of cancer cells.     

PPARγ potentiates anticancer effects of gemcitabine on human pancreatic cancer cells

In order to improve the prognosis of patients with unresectable pancreatic cancer, there is an urgent need for enhancement of the anticancer effect of gemcitabine (Gem), a first-line drug for the disease. Here, we demonstrated that ligands for peroxisome proliferator-activated receptor γ (PPARγ) such as pioglitazone (Pio) and rosiglitazone potentiated the cytotoxic action of Gem on human pancreatic cancer cells in a dosage-dependent manner. Notably, the synergistic effect was PPARγ-dependent, since the effect was augmented by PPARγ overexpression and was attenuated by both a PPARγ inhibitor (GW9662) and PPARγ-specific siRNA. To further increase the collaborative effect, the histone deacetylase (HDAC) inhibitor valproic acid (VPA), a known potentiator for PPARγ function, was added to the combinatorial treatment, robustly inducing apoptosis mediated by highly expressed death receptors, including Fas/CD95 and DR5. In xenograft tumor experiments in nude mice, Gem plus Pio significantly suppressed tumor growth as compared with the control treatment, while Gem-only treatment did not. Triple treatment with Gem, Pio, and VPA failed to demonstrate a significant antitumor effect when compared with Gem plus Pio in the current setting. Considered together, Gem plus PPARγ ligands, including Pio, may have therapeutic advantage in the treatment of advanced pancreatic cancer. Since Pio is widely used in the treatment of diabetes mellitus, it may become a feasible partner of Gem-based chemotherapy, fine-tuning the strength of the therapy in a dosage-dependent fashion.     

PKCα-induced drug resistance in pancreatic cancer cells is associated with transforming growth factor-β1

Background

Drug resistance remains a great challenge in the treatment of pancreatic cancer. The goal of this study was to determine whether TGF-β1 is associated with drug resistance in pancreatic cancer.

Methods

Pancreatic cancer BxPC3 cells were stably transfected with TGF-β1 cDNA. Cellular morphology and cell cycle were determined and the suppressive subtracted hybridization (SSH) assay was performed to identify differentially expressed genes induced by TGF-β1. Western blotting and immunohistochemistry were used to detect expression of TGF-β1-related genes in the cells and tissue samples. After that, the cells were further treated with an anti-cancer drug (e.g., cisplatin) after pre-incubated with the recombinant TGF-β1 plus PKCα inhibitor Gö6976. TGF-β1 type II receptor, TβRII was also knocked down using TβRII siRNA to assess the effects of these drugs in the cells. Cell viability was assessed by MTT assay.

Results

Overexpression of TGF-β1 leads to a markedly increased invasion potential but a reduced growth rate in BxPC3 cells. Recombinant TGF-β1 protein increases expression of PKCα in BxPC3 cells, a result that we confirmed by SSH. Moreover, TGF-β1 reduced the sensitivity of BxPC3 cells to cisplatin treatment, and this was mediated by upregulation of PKCα. However, blockage of PKCα with Gö6976 and TβRII with siRNA reversed the resistance of BxPC3 cells to gemcitabine, even in the presence of TGF-β1. Immunohistochemical data show that pancreatic cancers overexpress TGF-β1 and P-gp relative to normal tissues. In addition, TGF-β1 expression is associated with P-gp and membranous PKCα expression in pancreatic cancer.

Conclusions

TGF-β1-induced drug resistance in pancreatic cancer cells was associated with PKCα expression. The PKCα inhibitor Gö6976 could be a promising agent to sensitize pancreatic cancer cells to chemotherapy.     

Serum levels of IL-6 and IL-1β can predict the efficacy of gemcitabine in patients with advanced pancreatic cancer

Background:

With this study, we sought to characterise the impact of pro-inflammatory cytokines on the outcomes of gemcitabine monotherapy (GEM) in patients with pancreatic cancer (PC).

Methods:

Treatment-naive patients with advanced PC and no obvious infections were eligible for enrolment. All of the patients were scheduled to undergo systemic chemotherapy. Serum pro-inflammatory cytokines were measured using an electro-chemiluminescence assay method before chemotherapy. High cytokine levels were defined as values greater than the median. Clinical data were collected prospectively.

Results:

Sixty patients who received GEM were included in the analysis. High IL-6 and IL-1β levels were poor prognostic factors for overall survival in a multivariate analysis (P=0.011 and P=0.048, respectively). Patients with both a high IL-6 level and a high IL-1β level exhibited shortened overall and progression-free survival, a reduction in the tumour control rate, and a high dose intensity of GEM compared with patients with low levels of both IL-6 and IL-1β.

Conclusion:

The serum levels of IL-6 and IL-1β predict the efficacy of GEM in patients with advanced PC.     

Supraadditive effect of 2′,2′-difluorodeoxycytidine (gemcitabine) in combination with oxaliplatin in human cancer cell lines

PURPOSE: This study assessed the cytotoxic effects of the nucleoside analog gemcitabine in combination with the diaminocyclohexane platinum compound oxaliplatin. METHODS: Growth inhibition studies were performed using the human CEM leukemia cell line and the colon-cancer cell lines HCT 116 and Colo 320 DM. Gemcitabine-oxaliplatin combinations were compared with gemcitabine-cisplatin combinations in the same cell lines using similar experimental settings. Cells were exposed for 2 h to gemcitabine and then for 24 h to oxaliplatin or cisplatin, and vice versa. RESULTS: The 50% inhibitory concentrations (IC50 values) in single-drug experiments using 2 h of exposure to gemcitabine and 24 h of exposure to oxaliplatin or cisplatin were, respectively, 89 pM, 11.1 microM, and 10.3 microM for CEM cells; 46 pM, 10.2 microM, and 2.7 microM for HCT 116 cells; and 102 pM, 4.6 microM, and 8.6 microM for Colo 320 DM cells. Gemcitabine-oxaliplatin combinations displayed supraadditive effects in human leukemia and colon-cancer cell lines. The sequence of gemcitabine followed by oxaliplatin was more effective than the opposite sequence in HCT 116 and Colo 320 DM colon-cancer cell lines, whereas the sequence of oxaliplatin followed by gemcitabine yielded to synergistic effects in CEM cells. The cytotoxic effects of gemcitabine-oxaliplatin combinations were better than (HCT 116 cells) or equal to (CEM and Colo 320 DM cells) those of gemcitabine-cisplatin combinations. CONCLUSION: Our data show that the combination of gemcitabine with oxaliplatin exerts potent antiproliferative effects in human leukemia and colon cancer cells, warranting further investigations in the framework of phase I-II trials as an alternative for the treatment of solid malignancies.     

Phase II trial of S-1 in combination with gemcitabine for chemo-naïve patients with locally advanced or metastatic pancreatic cancer

Background

We performed a phase II study of combination chemotherapy with S-1 plus gemcitabine for treating chemo-naïve patients with unresectable pancreatic cancer to evaluate the efficacy and toxicity.

Patients and methods

Patients with histologically confirmed unresectable pancreatic cancer were eligible. The treatment consisted of S-1 (40 mg/m² p.o. b.i.d. from D1 to 14) and gemcitabine (1,250 mg/m² on D1 and 8), repeated every 3 weeks.

Results

Thirty-two patients were enrolled between March 2005 and December 2007. No complete response was observed and a partial response was observed in 14 patients (44.0%), stable disease in eight patients (25.0%), and progressive disease in eight patients (25.0%). The median time to progression was 4.92 months (95% CI: 4.16–5.67 months), and the median overall survival was 7.89 months (95% CI: 5.96–9.82 months). The survival duration was significantly longer for the patients with a good performance status compared with that of the patients with a poor performance status. The major toxicities were grade 3–4 neutropenia (9, 28.1%), grade 3/4 thrombocytopenia (5, 15.6%), and grade 3 diarrhea (5, 15.6%).

Conclusion

The combination chemotherapy of S-1 and gemcitabine showed promising antitumor activity and manageable toxicities, and especially for the good performance status patients with unresectable pancreatic cancer.     

MicroRNA-21 induces resistance to the anti-tumour effect of interferon-α/5-fluorouracil in hepatocellular carcinoma cells

Background:

We reported recently the clinical efficiency of interferon (IFN)-α/5-fluorouracil (5-FU) combination therapy in advanced hepatocellular carcinoma (HCC). However, prediction of the response to the combination therapy remains unsatisfactory. The aim of this study was to investigate the anti-tumour effects of microRNA (miR)-21 on the sensitivity of HCC cells to IFN-α/5-FU and whether miR-21 can be used as a predictor of the response to such therapy in HCC.

Methods:

Changes in the sensitivity of HCC cells (PLC/PRF/5 and HepG2) to IFN-α/5-FU were examined after transfection with pre-miR-21 or anti-miR-21. The correlation between miR-21 expression level, evaluated by qRT–PCR, and response to the therapy was also investigated in clinical HCC specimens.

Results:

Hepatocellular carcinoma cells transfected with pre-miR-21 were significantly resistant to IFN-α/5-FU. Annexin V assay showed that the percentage of apoptotic cells was significantly lower in cells transfected with pre-miR-21 than control cells. Transfection of anti-miR-21 rendered HCC cells sensitive to IFN-α/5-FU, and such sensitivity was weakened by transfection of siRNAs of target molecules, PETN and PDCD4. miR-21 expression in clinical HCC specimens was significantly associated with the clinical response to the IFN-α/5-FU combination therapy and survival rate.

Conclusions:

The miR-21 in HCC cell lines and clinical HCC samples is a significant modulator of the anti-tumour effect of IFN-α and 5-FU. This suggests that miR-21 is a potentially suitable marker for the prediction of the clinical response to the IFN-α/5-FU combination therapy.     

Anti-apoptotic effect of claudin-1 on TNF-α-induced apoptosis in human breast cancer MCF-7 cells

Accumulating evidence reveals that aberrant expression of claudins manifests in various tumors; however, their biological functions are poorly understood. Here, we report on the elevated expression of claudin-1 in human breast cancer MCF-7 cells under tumor necrosis factor (TNF)-?? treatment. Interestingly, the increased expression of claudin-1 contributes to an anti-apoptotic role in TNF-??-induced apoptosis. In line with this, upon TNF-?? stimulus, downregulation of claudin-1 by siRNA knockdown results in a significant increase in cleavage of caspase-8 and poly (ADP-ribose) polymerase, a decrease of cyclinD1 expression, and DNA fragmentation. Consistently, TdT-mediated dUTP nick end labeling assay also shows that loss of claudin-1 increases the susceptibility of MCF-7 cells to TNF-??-induced apoptosis. However, there is no obvious effect on the expression of Bax and p53 after the treatment aforementioned. In addition, TNF-?? increases the amount of claudin-1 and the cytoplasmic accumulation of ??-catenin, while claudin-1 siRNA increases the amount of ??-catenin in the cell membrane as well as the amount of E-cadherin in the cytoplasm. In conclusion, our data reveal a novel role of claudin-1 in regulating apoptosis in MCF-7 cells.     

Phase II trial of two-weekly gemcitabine in patients with advanced biliary?tract cancer

Background:Patients with advanced biliary tract carcinoma facea dismal prognosis as no effective palliative therapy has been defined. Theaim of the present phase II investigation was to evaluate the therapeuticefficacy and tolerance of a two-weekly high-dose gemcitabine regimen in thispatient population. Patients and methods:Thirty-two consecutive patients with locallyunresectable or metastatic biliary tract cancer were enrolled in thismulticenter phase II trial. Treatment consisted of gemcitabine 2200mg/m² given as a 30-min intravenous infusion every two weeks fora duration of six months unless there was prior evidence of progressivedisease. Results:After a median number of 12 treatment courses, 7 of32 (22%) patients had a partial response that lasted for a medianduration of 6.0 months (range 3.5–10.0). Fourteen additional patients(44%) had stable disease, whereas eleven patients (34%)progressed despite therapy. The median time to progression was 5.6 months(range 1.8–13.0); median survival time was 11.5 months (range3.0–24.0), and the probability of surviving beyond 12 months was44%. The tolerance of treatment was remarkable with only two patientseach experiencing grade 3 leukocytopenia, granulocytopenia and/orthrombocytopenia, and one patient had grade 3 anaemia. Similarly,nonhaematologic side effects were infrequent, and generally mild tomoderate. Conclusions:Two-weekly high-dose gemcitabine seems to representa potentially effective, safe and well-tolerated regimen for the palliativetreatment of patients with advanced biliary tract cancer.     

Mechanisms underlying gemcitabine resistance in pancreatic cancer and sensitisation by the iMiD™ lenalidomide

Gemcitabine is currently the leading therapeutic for pancreatic cancer treatment, despite growing resistance. Studying the mechanisms that underlie gemcitabine resistance and discovery of agents that increase the tumour sensitivity to gemcitabine, is therefore desirable. The thalidomide analogue lenalidomide has been approved for use in multiple myeloma in combination with dexamethasone. Although it is primarily immunomodulatory, it also has direct effects on tumours. We investigated the sensitivity of three pancreatic cell lines PANC-1, MIA-PaCa-2 and BxPC-3 to gemcitabine. We observed that PANC-1 cells display most resistance to gemcitabine and MIA-PaCa-2 are most sensitive. Western blot analysis revealed that PANC-1 exhibits high phosphorylated extracellular signal-regulated kinase (pERK) expression, whereas MIA-PaCa-2 displays low expression. Combining gemcitabine and lenalidomide reduced the IC(50) of gemcitabine up to 40% (p<0.05). Western blot analysis showed lenalidomide significantly reduced pERK expression in all cell lines (p<0.05). It was hypothesised that gemcitabine sensitivity could be increased through combination with a pERK-reducing agent. The mitogen-activated kinase (MEK) specific inhibitor U0126 was used on PANC-1 cells to restore gemcitabine sensitivity. U0126 significantly increased cell killing by gemcitabine from 30% to 60% (p<0.001). Sensitive MIA-PaCa-2 cells were transfected with a constitutively active MEK mutant to reduce gemcitabine sensitivity. Transfection resulted in a significant reduction in cell killing by gemcitabine from 54-16% (p<0.05). These results provide evidence that ERK activity underlies sensitivity to gemcitabine and that addition of an agent that reduces this activity, such as lenalidomide, enhances gemcitabine efficacy. In conclusion, these results provide an understanding of gemcitabine resistance and could be used to predict successful combination therapies.     

A feasibility study of carboplatin with fixed dose of gemcitabine in ‘unfit’ patients with advanced bladder cancer

For the purpose of a subsequent phase II/III European Organization for Research and Treatment of Cancer (EORTC) trial, a gemcitabine/carboplatin feasibility study in ‘unfit’ patients with advanced urothelial cell cancer was conducted. Gemcitabine was given at 1000 mg/m² days 1 and 8 with carboplatin (area under the curve (AUC) 4.5 or 5) day 1 every 21 days. 16 patients were treated, median age 68 years (47–75) years, performance status (PS) 0/1/2 in 3/10/3 patients. Creatinine clearance was >1 ml/s in 3 patients, 0.5–1 ml/s in 9 and <0.5 ml/s in 4 patients. Half of the patients had visceral disease. Median number of cycles given was 4 (range 2–6), for a total of 69 cycles. The first 8 patients received 33 cycles using a carboplatin AUC of 5. World Health Organization (WHO) grade 3-4 toxicity was: haemoglobin 5 patients, platelets 6 patients, neutrophils 5 patients and febrile neutropenia 2 patients. In view of this haematological toxicity in subsequent patients, the carboplatin AUC was decreased to 4.5. At this dose level, 8 patients received 36 cycles. WHO grade 3-4 toxicity was: anaemia 1 patient, platelets 4 patients, neutrophils 4 patients with no febrile neutropenia. Thus, this dose level was regarded to be feasible. For the 16 evaluable patients, overall response rate was 44%, (1 complete response (CR), 6 partial response (PR)). In conclusion, the combination of gemcitabine with carboplatin at an AUC of 4.5 appears to be an active and well tolerated regimen with acceptable toxicity in this unfit patient population. Based on these data, a randomised trial in the framework of the EORTC-Genitourinary (GU) group of gemcitabine/carboplatin versus carboplatin/methotrexate/vinblastine (MCAVI) is ongoing.     

α-Catulin knockdown induces senescence in cancer cells

Cellular senescence functions as a tumor suppressor that protects against cancer progression. α-Catulin, an α-catenin-related protein, is reported to have tumorigenic potential because it regulates the nuclear factor-κB (NF-κB) pathway, but little is known about its clinical relevance and the mechanism through which it regulates cancer progression. Here, we found that α-catulin mRNA levels were significantly upregulated in cancer cell lines and clinical oral squamous cell carcinomas, which positively correlated with tumor size (P=0.001) and American Joint Committee on Cancer (AJCC) stage (P=0.004). α-Catulin knockdown in the OC2 and A549 cancer cell lines dramatically decreased cell proliferation and contributed to cellular senescence, and inhibited OC2 xenograft growth. Mechanistic dissection showed that α-catulin depletion strongly induced the DNA-damage response (DDR) in both cell lines, via a p53/p21-dependent pathway in A549 cells, but a p53/p21-independent pathway in OC2 cells carrying mutant p53. Global gene expression analysis revealed that α-catulin knockdown altered cell-cycle regulation and DDR pathways at the presenescent stage as well as significantly downregulate several crucial genes related to mitotic chromosome condensation, DDR and DNA repair systems, which suggests that its depletion-induced cellular senescence might be caused by chromosome condensation failures, severe DNA damage and impaired DNA repair ability. Our study provides evidence that α-catulin promotes tumor growth by preventing cellular senescence and suggests that downregulating α-catulin may be a promising therapeutic approach for cancer treatment.     

Combined antitumor effect of γ-secretase inhibitor and ABT-737 in Notch-expressing non-small cell lung cancer

Background

Inhibition of Notch by γ-secretase inhibitor (GSI) has been shown to have an antitumor effect in Notch-expressing non-small cell lung cancer (NSCLC) and to induce apoptosis through modulation of Bcl-2 family proteins. In particular, Bim, a BH3-only member of the Bcl-2 family of proteins, has an important role in the induction of apoptosis in NSCLC when cells are treated with GSI. ABT-737, a BH3-only mimetic, targets the pro-survival Bcl-2 family and also induces apoptosis.

Methods

The Notch-expressing NSCLC cell lines H460, A549, H1793, and HCC2429 were used. The combined antitumor effect of GSI and ABT-737 was evaluated using the MTT proliferation assay in vitro and in xenograft mouse models. The expression of the Notch pathway and Bcl-2 family was analyzed using Western blotting analysis when cells were treated with a single drug treatment or a combination treatment.

Results

GSI XX or ABT-737 alone inhibited cell proliferation in a dose-dependent manner, and combination drug treatment showed a synergistic antitumor effect in vitro. In vivo, this drug combination significantly suppressed tumor proliferation compared to the single drug treatment. Phospho-Bcl-2 was downregulated and Bax was upregulated by both the single and combination drug treatments. Bim was induced by a single drug treatment and was enhanced by combination treatment. Combination treatment-induced apoptosis was decreased by Bim inhibition, suggesting that the antitumor effect of the drug combination was dependent on Bim.

Conclusion

Based on our data, we propose that the combination treatment is a promising strategy for NSCLC therapy.