Bortezomib induces protective autophagy through AMP-activated protein kinase activation in cultured pancreatic and colorectal cancer cells

Background

Bortezomib, a selective and potent inhibitor of the proteasome, has demonstrated broad anti-tumor activities in many malignancies. In the current study, we aimed to understand the potential resistance factor of bortezomib in cultured pancreatic and colorectal cancer cells.

Results

We observed that bortezomib-induced protective autophagy in cultured PANC-1 pancreatic cancer cells and HT-29 colorectal cancer cells. Inhibition of autophagy by 3-methyladenine (3-MA) and chloroquine enhanced bortezomib-induced apoptosis and cytotoxicity in both PANC-1 and HT-29 cells. Activation of AMP-activated protein kinase (AMPK) was required for bortezomib-induced autophagy induction in PANC-1 and HT-29 cells, and AMPK inhibition by its inhibitor compound C (CC) or RNAi-depletion suppressed bortezomib-induced autophagy, while dramatically enhancing cancer cell apoptosis/cytotoxicity. Meanwhile, significant AMPK activation and autophagy induction were observed after bortezomib stimulation in primary cultured pancreatic cancer cells derived from a patient’s tumor tissue. Both CC and 3-MA facilitated bortezomib-induced cytotoxicity in primary cultured pancreatic cancer cells.

Conclusions

In conclusion, our data here suggest that bortezomib induces protective autophagy in pancreatic and colorectal cancer cells through activating AMPK-Ulk1 signalings. AMPK or autophagy inhibitors could be developed as an adjunct or chemo-sensitizer for bortezomib.     

CIP2A is a target of bortezomib in human triple negative breast cancer cells

Introduction

Triple negative breast cancer (TNBC) is very aggressive and currently has no specific therapeutic targets, such as hormone receptors or human epidermal growth factor receptor type 2 (HER2); therefore, prognosis is poor. Bortezomib, a proteasome inhibitor, may exert efficacy in TNBC through its multiple cellular effects. Here, we tested the efficacy of bortezomib and examined the drug mechanism in breast cancer cells.

Methods

Five breast cancer cell lines: TNBC HCC-1937, MDA-MB-231, and MDA-MB-468; HER2-overexpressing MDA-MB-453; and estrogen receptor positive MCF-7 were used for in vitro studies. Apoptosis was examined by both flow cytometry and Western Blot. Signal transduction pathways in cells were assessed by Western Blot. Gene silencing was done by small interfering RNA (siRNA). In vivo efficacy of bortezomib was tested in nude mice with breast cancer xenografts. Immunohistochemical study was performed on tumor tissues from patients with TNBC.

Results

Bortezomib induced significant apoptosis, which was independent of its proteasome inhibition, in the three TNBC cell lines, but not in MDA-MB-453 or MCF-7 cells. Furthermore, cancerous inhibitor of protein phosphatase 2A (CIP2A), a cellular inhibitor of protein phosphatase 2A (PP2A), mediated the apoptotic effect of bortezomib. We showed that bortezomib inhibited CIP2A in association with p-Akt downregulation in a dose- and time-dependent manner in all sensitive TNBC cells, whereas no alterations in CIP2A expression and p-Akt were noted in bortezomib-resistant cells. Overexpression of CIP2A upregulated p-Akt and protected MDA-MB-231 and MDA-MB-468 cells from bortezomib-induced apoptosis, whereas silencing CIP2A by siRNA overcame the resistance to bortezomib-induced apoptosis in MCF-7 cells. In addition, bortezomib downregulated CIP2A mRNA but did not affect the degradation of CIP2A protein. Furthermore, bortezomib exerted in vivo antitumor activity in HCC-1937 xenografted tumors, but not in MCF-7 tumors. Bortezomib downregulated CIP2A expression in the HCC-1937 tumors but not in the MCF-7 tumors. Importantly, CIP2A expression is readily detectable in tumor samples from TNBC patients.

Conclusions

CIP2A is a major determinant mediating bortezomib-induced apoptosis in TNBC cells. CIP2A may thus be a potential therapeutic target in TNBC.     

Preventing the autophagic survival response by inhibition of calpain enhances the cytotoxic activity of bortezomib in vitro and in vivo

Purpose

Bortezomib, a first-generation proteasome inhibitor, induces an endoplasmic reticulum (ER) stress response, which ultimately leads to dysregulation of intracellular Ca²⁺ and apoptotic cell death. This study investigated the role of the Ca²⁺-dependent enzyme, calpain, in bortezomib cytotoxicity. A novel therapeutic combination was evaluated in which HIV protease inhibitors were used to block calpain activity and enhance bortezomib cytotoxicity in myeloma cells in vitro and in vivo.

Methods

Bortezomib-mediated cell death was examined using assays for apoptosis (Annexin V staining), total cell death (trypan blue exclusion), and growth inhibition (MTT). The effects of calpain on bortezomib-induced cytotoxicity were investigated using siRNA knockdown or pharmaceutical inhibitors. Enzyme activity assays and immunofluorescence analysis were used to identify mechanistic effects.

Results

Inhibition of the Ca²⁺-dependent cysteine protease calpain, either by pharmacologic or genetic means, enhances or accelerates bortezomib-induced myeloma cell death. The increase in cell death is not associated with an increase in caspase activity, nor is there evidence of greater inhibition of proteasome activity, suggesting an alternate, calpain-regulated mechanism of bortezomib-induced cell death. Bortezomib initiates an autophagic response in myeloma cells associated with cell survival. Inhibition of calpain subverts the cytoprotective function of autophagy leading to increased bortezomib-mediated cell death. Combination therapy with bortezomib and the calpain-blocking HIV protease inhibitor, nelfinavir, reversed bortezomib resistance and induced near-complete tumor regressions in an SCID mouse xenograft model of myeloma.     

C6 ceramide potentiates curcumin-induced cell death and apoptosis in melanoma cell lines in vitro

Purpose

The majority of metastatic melanomas are resistant to diverse chemotherapeutic agents, and long-term survival for patients with melanoma who have metastatic disease is dismal. Consequently, the search for novel anti-melanoma agents is urgent. Here, we evaluate the potential effects of C6 ceramide to sensitize melanoma cell lines (B16 and WM-115 cells) to curcumin-induced cell death.

Methods

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to test melanoma cell viability in vitro. Hoechst 33342 fluorescence and Histone DNA ELISA was used to evaluate melanoma cell apoptosis. Apoptosis-associated proteins in melanoma cells after treatments were measured by Western blot.

Results

C6 ceramide promotes curcumin-induced cell death and apoptosis in B16 and WM-115 melanoma cell lines. Curcumin itself promotes pro-apoptosis protein Caspase 3 and Caspase 9 cleavage and anti-apoptosis protein Bcl-XL and X-IAP degradation, and combination of C6 ceramide with curcumin dramatically enhances it. Caspase inhibitors largely inhibit C6-ceramide plus curcumin induced cell death and apoptosis.

Conclusion

We suggest that C6 ceramide sensitizes melanoma cell to curcumin induced cell death and apoptosis in vitro, which is due to, at least in part, the augment of mitochondria apoptosis pathway. Combining C6 ceramide with traditional chemotherapy drugs such as curcumin may have potential to be used as a new therapeutic intervention against melanoma.     

Resistance to the antiproliferative effect induced by a short-chain ceramide is associated with an increase of glucosylceramide synthase,P-glycoprotein,and multidrug-resistance gene-1 in cervical cancer cells

Purpose

Ceramide is glycosylated to glucosylceramide or lactosylceramide, and this glycosylation is a novel multidrug-resistance (MDR) mechanism. In this work, a short-chain ceramide (C6), lactosylceramide (LacCer), and an inhibitor of ceramide glycosylation (d-threo-1-phenyl-2-decanoylamino-3-1-propanol, PDMP) were evaluated on the proliferation of cervical cancer cells. The participation of glucosylceramide synthase (GCS), P-glycoprotein (P-gp), and multidrug-resistance gene-1 (MDR-1) in the resistance to the antiproliferative effect induced by C6 was also evaluated.

Methods

Cell proliferation was determined by crystal violet staining. GCS and MDR-1 mRNA expression was evaluated by real-time RT-PCR assay. GCS and P-gp protein expressions, as well as Rhodamine 123 uptake, which is a functional test for P-gp efflux activity, were determined by flow cytometry.

Results

C6 inhibited proliferation of CaLo and CasKi cells with an IC₅₀ of 2.5 μM; however, 50 % proliferation of ViBo cells was inhibited with 10 μM. LacCer increased the proliferation of all cells. When cells were treated with PDMP plus C6, no additional effect on antiproliferation induced by C6 was observed in CaLo and CasKi cells; however, proliferation diminished in comparison with C6 alone in ViBo cells. C6 increased GCS and MDR-1 expression in all cells, as well as P-gp expression in CasKi cells.

Conclusions

Cells that have more capacity to glycosylate ceramide and express a higher level of GCS, MDR-1, and P-gp, are more resistant to the antiproliferative effect induced by C6.     

Deoxycholate promotes survival of breast cancer cells by reducing the level of pro-apoptotic ceramide

Introduction

At physiologic concentration in serum, the bile acid sodium deoxycholate (DC) induces survival and migration of breast cancer cells. Here we provide evidence of a novel mechanism by which DC reduces apoptosis that is induced by the sphingolipid ceramide in breast cancer cells.

Methods

Murine mammacarcinoma 4T1 cells were used in vitro to determine apoptosis and alteration of sphingolipid metabolism by DC, and in vivo to quantify the effect of DC on metastasis.

Results

We found that DC increased the number of intestinal metastases generated from 4T1 cell tumors grafted into the fat pad. The metastatic nodes contained slowly dividing cancer cells in immediate vicinity of newly formed blood vessels. These cells were positive for CD44, a marker that has been suggested to be expressed on breast cancer stem cells. In culture, a subpopulation (3 ± 1%) of slowly dividing, CD44⁺ cells gave rise to rapidly dividing, CD44^- cells. DC promoted survival of CD44⁺ cells, which was concurrent with reduced levels of activated caspase 3 and ceramide, a sphingolipid inducing apoptosis in 4T1 cells. Z-guggulsterone, an antagonist of the farnesoid-X-receptor, obliterated this anti-apoptotic effect, indicating that DC increased cell survival via farnesoid-X-receptor. DC also increased the gene expression of the vascular endothelial growth factor receptor 2 (Flk-1), suggesting that DC enhanced the initial growth of secondary tumors adjacent to blood vessels. The Flk-1 antagonist SU5416 obliterated the reduction of ceramide and apoptosis by DC, indicating that enhanced cell survival is due to Flk-1-induced reduction in ceramide.

Conclusions

Our findings show, for the first time, that DC is a natural tumor promoter by elevating Flk-1 and decreasing ceramide-mediated apoptosis of breast cancer progenitor cells. Reducing the level or effect of serum DC and elevating ceramide in breast cancer progenitor cells by treatment with Z-guggulsterone and/or vascular endothelial growth factor receptor 2/Flk-1 antagonists may thus be a promising strategy to reduce breast cancer metastasis.     

Potential role of acid ceramidase in conversion of cytostatic to cytotoxic end-point in pancreatic cancer cells

Purpose

Acid ceramidase (AC) occupies an important place in the control of cancer cell proliferation. We tested the influence of AC inhibition on the effects of PSC 833, a P-glycoprotein antagonist with potent ceramide-generating capacity, to determine whether AC could be a therapeutic target in pancreatic cancer.

Methods

Ceramide metabolism was followed using ³H-palmitate, and molecular species were determined by mass spectroscopy. Apoptosis was measured by DNA fragmentation, autophagy by acridine orange staining, and cell cycle was assessed by flow cytometry and RB phosphorylation. AC was measured in intact cells using fluorescent substrate.

Results

Exposure of human PANC-1 or MIA-PaCa-2 cells to PSC 833 promoted increases in de novo (dihydro)ceramides, (dihydro)glucosylceramides, and (dihydro)sphingomyelins, demarking ceramide generation and robust metabolism. Despite the multifold increases in (dihydro)ceramide levels, cells were refractory to PSC 833. However, PSC 833 produced a dose-dependent decrease in DNA synthesis and dose- and time-dependent decreases in RB phosphorylation, consistent with cell cycle arrest as demonstrated at G1. Cytostatic effects of PSC 833 were converted to cytotoxic end-point by acid ceramidase inhibition. Cytotoxicity was accompanied by formation of acridine orange-stained acidic vesicles and an increase in LC3 expression, indicative of autophagic response. Cell death was not reversed by preexposure to myriocin, which blocks PSC 833-induced ceramide generation.

Conclusion

Although the role of ceramide in end-point cytotoxicity is unclear, our results suggest that acid ceramidase is a viable target in pancreatic cancer. We propose that AC inhibition will be effective in concert with other anticancer therapies.     

The role of K-Ras gene mutation in TRAIL-induced apoptosis in pancreatic and lung cancer cell lines

Purpose

Pancreatic ductal and lung adenocarcinomas are the most common and prevalent types of human neoplasms with a greater than 80% mortality rate. The poor prognosis of both these cancers are likely due to the absence of valid approaches for early detection, the frequency of its metastases at the time of diagnosis, frequent recurrence after surgery, and poor responsiveness to chemotherapy. Most notably, the early development of pancreatic intraepithelial neoplasia and lung lesions is suggested to be the result of a mutation in the K-ras (G12D) oncogene. Tumor necrosis factor-related-apoptosis-inducing-ligand (TRAIL) has been shown to have great potential for the treatment of most human tumor cells, while leaving normal cells unharmed. However, some cancers show resistance to TRAIL treatment, leaving a gap in the understanding of its exact etiology.

Methods

TRAIL-induced resistance to cell death was investigated in pancreatic and lung cancer cell lines. Cell survival was determined by SRB and apoptosis by ELISA-based cell death assay. Activation of bid and caspases were evaluated by Western blotting.

Results

Our study demonstrated that TRAIL significantly suppressed cell survival, by inducing apoptosis in a dose-dependent manner, in the pancreatic cancer BxPC-3 (wild type G12) and lung cancer A549 (G12S) cell lines. In contrast, Panc-1 pancreatic and SK-LU-1 lung cancer cell lines, which have a mutated (G12D) K-ras genotype, were resistant to the actions of TRAIL.

Conclusions

This study demonstrates an association between TRAIL resistance to apoptosis in human pancreatic and lung cancer cell lines and G12D K-ras¹² mutation.     

Resveratrol induces apoptosis of pancreatic cancers cells by inhibiting miR-21 regulation of BCL-2 expression

Objective

Resveratrol is an edible polyphenolic phytoalexin present in different plant species and plays important role in inhibiting proliferation and inducing apoptosis of pancreatic cancer cells. In this paper, the mechanism of resveratrol on PANC-1, CFPAC-1 and MIA Paca-2 cells apoptosis was examined.

Methods

We first evaluated the effect of resveratrol on viability of PANC-1, CFPAC-1 and MIA Paca-2 cells using MTT assay. Next, we performed real-time PCR to assess the effect of resveratrol on miR-21 expression. We also used Western blot to measure BCL-2 protein levels after down-regulation of miR-21 expression. Finally, we evaluated the effect of miR-21 on resveratrol-induced anti-tumor activity using miR-21 mimic.

Results

Resveratrol induced a significant inhibition of PANC-1, CFPAC-1 and MIA Paca-2 cells viability in a dose-dependent manner. Resveratrol also decreased the expression of miR-21. Besides, down-regulation of miR-21 expression can inhibit BCL-2 expression in PANC-1, CFPAC-1 and MIA Paca-2 cells. Over-expression of miR-21 expression can reverse down-regulation of BCL-2 expression and apoptosis induced by resveratrol.

Conclusions

In this study, we demonstrated that the effect of resveratrol on apoptosis is due to inhibiting miR-21 regulation of BCL-2 expression.     

The interaction of bortezomib with multidrug transporters: implications for therapeutic applications in advanced multiple myeloma and other neoplasias

Purpose

Bortezomib is an important agent in multiple myeloma treatment, but resistance in cell lines and patients has been described. The main mechanisms of resistance described in cancer fall into one of two categories, pharmacokinetic resistance (PK), e.g. over expression of drug efflux pumps and pharmacodynamic resistance, e.g. apoptosis resistance or altered survival pathways, where the agent reaches an appropriate concentration, but this fails to propagate an appropriate cell death response. Of the known pump mechanisms, P-glycoprotein (P-gp) is the best studied and considered to be the most important in contributing to general PK drug resistance. Resistance to bortezomib is multifactorial and there are conflicting indications that cellular overexpression of P-gp may contribute to resistance agent. Hence, better characterization of the interactions of this drug with classical resistance mechanisms should identify improved treatment applications.

Methods

Cell lines with different P-gp expression levels were used to determine the relationship between bortezomib and P-gp. Coculture system with stromal cells was used to determine the effect of the local microenvironment on the bortezomib–elacridar combination. To further assess P-gp function, intracellular accumulation of P-gp probe rhodamine-123 was utilised.

Results

In the present study, we show that bortezomib is a substrate for P-gp, but not for the other drug efflux transporters. Bortezomib activity is affected by P-gp expression and conversely, the expression of P-gp affect bortezomib’s ability to act as a P-gp substrate. The local microenvironment did not alter the cellular response to bortezomib. We also demonstrate that bortezomib directly affects the expression and function of P-gp.

Conclusions

Our findings strongly support a role for P-gp in bortezomib resistance and, therefore, suggest that combination of a P-gp inhibitor and bortezomib in P-gp positive myeloma would be a reasonable treatment combination to extend efficacy of this important drug.     

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.     

Gamma-secretase inhibitor enhances the cytotoxic effect of bortezomib in multiple myeloma

Background

Targeting of Notch signaling with γ-secretase inhibitors (GSIs) has been considered a promising strategy for the treatment of hematological malignancies including multiple myeloma (MM). Here we investigated whether the cytotoxic effect of bortezomib, an agent commonly used in MM, could be enhanced by the addition of a GSI.

Methods

MM cells were treated with GSI, bortezomib or the combination thereof. Apoptosis of MM cells, proteasome activity and Notch signaling activation were determined. The effect of the drug combination was also evaluated in MM cells transfected with the active domain of Notch-1.

Results

Using MM cell lines and primary MM cells isolated from the bone marrow of patients with MM we found a strong synergistic effect of bortezomib in combination with one of the GSIs studied. We next investigated the mechanism underlying this synergistic effect and determined that the effect of the drug combination was mainly dependent on the ability of the selected GSI to inhibit proteasome activity in MM cells.

Conclusion

Our study demonstrates that selected GSIs that inhibit proteasome activity may be successfully used in combination with bortezomib enhancing its anti-MM effect.     

Perifosine inhibits S6K1–Gli1 signaling and enhances gemcitabine-induced anti-pancreatic cancer efficiency

Purpose

The pancreatic cancer has extremely low overall 5-year survival, and gemcitabine is the only approved single agent for pancreatic cancer treatment.

Methods

In the present study, we investigated the potential effect of perifosine, a novel Akt inhibitor on gemcitabine-induced anti-pancreatic cancer effect both in vivo and in vitro.

Results

We showed that sub-cytotoxic low concentration of perifosine dramatically enhanced gemcitabine-induced cytotoxicity in cultured pancreatic cancer cells. Perifosine inhibited Akt–mammalian target of rapamycin and Erk–mitogen-activated protein kinase activation in pancreatic cancer cells. Meanwhile, perifosine suppressed the hedgehog signaling, as it inhibited glioma-associated oncogenes (Gli) 1 activation and decreased its target protein patched 1 (PTCH1) expression. Our data demonstrated that perifosine blocked p70S6K1 (S6K1) activation, thus disrupting S6K1–Gli1 association and subsequent Gli1 activation. The reduction of S6K1 or Gli1 expression by target siRNAs inhibited PTCH1 expression and enhanced gemcitabine-induced cytotoxicity in pancreatic cancer cells. Significantly, perifosine dramatically enhanced gemcitabine-mediated antitumor effect in a PANC-1 xenograft severe combined immunodeficiency mice model.

Conclusions

In summary, we conclude that perifosine sensitizes gemcitabine-mediated anti-pancreatic cancer efficiency through regulating multiple signaling pathways.     

Schwann cell autophagy induced by SAHA, 17-AAG, or clonazepam can reduce bortezomib-induced peripheral neuropathy

Background:

The proteasome inhibitor bortezomib has improved the survival of patients with multiple myeloma but bortezomib-induced peripheral neuropathy (BiPN) has emerged as a serious potential complication of this therapy. Animal studies suggest that bortezomib predominantly causes pathological changes in Schwann cells. A tractable system to evaluate combination drugs for use with bortezomib is essential to enable continuing clinical benefit from this drug.

Methods:

Rat schwannoma cells were pretreated with vincristine (VCR), histone deacetylase inhibitors, anticonvulsants, or a heat-shock protein 90 (HSP90) inhibitor. To then monitor aggresome formation as a result of proteasome inhibition and the activation of chaperone-mediated autophagy (CMA), we performed double-labelling immunofluorescent analyses of a cellular aggregation-prone protein marker.

Results:

Aggresome formation was interrupted by VCR, whereas combination treatments with bortezomib involving suberoylanilide hydroxamic acid, 17-allylamino-17-demethoxy-geldanamycin, or clonazepam appear to facilitate the disposal of unfolded proteins via CMA, inducing HSP70 and lysosome-associated membrane protein type 2A (LAMP-2A).

Conclusions:

This schwannoma model can be used to test BiPN-reducing drugs. The present data suggest that aggresome formation in Schwann cells is a possible mechanism of BiPN, and drugs that induce HSP70 or LAMP-2A have the potential to alleviate this complication. Combination clinical trials are warranted to confirm the relevance of these observations.     

Overexpression of AIB1 correlates inversely with E-cadherin expression in pancreatic adenocarcinoma and may promote lymph node metastasis

Background

It was reported that the nuclear receptor coactivator amplified in breast cancer1 (AIB1) could regulate cancer cell invasion and migration in a nuclear receptor signaling-independent manner. Meanwhile, the process of epithelial mesenchymal transition (EMT) is critical for tumor invasion and metastasis. The present study aimed to determine the role of AIB1 and EMT markers in human pancreatic adenocarcinoma.

Methods

AIB1, ZO-1, E-cadherin, vimentin, and N-cadherin protein expression in 76 pancreatic adenocarcinomas were assessed using immunohistochemistry and analyzed for clinicopathological significance.

Results

The frequency of AIB1 overexpression in pancreatic adenocarcinomas with lymph node metastasis is 68 % (19/28), which is significantly higher than in pancreatic adenocarcinomas without lymph node metastasis (42 %; 20/48). In addition, the frequency of low expression of E-cadherin in pancreatic carcinomas with lymph node metastasis (68 %; 19/28) was significantly higher than in tumors without lymph node metastasis (44 %; 21/48). Correlation analysis demonstrated that the overexpression of AIB1 was inversely correlated with low expression of E-cadherin in pancreatic adenocarcinomas.

Conclusion

Overexpression of AIB1 might promote invasion and metastasis of cancer cells and is associated with down-regulation of E-cadherin in pancreatic adenocarcinomas.     

Corticosteroid co-treatment induces resistance to chemotherapy in surgical resections, xenografts and established cell lines of pancreatic cancer

Background

Chemotherapy for pancreatic carcinoma often has severe side effects that limit its efficacy. The glucocorticoid (GC) dexamethasone (DEX) is frequently used as co-treatment to prevent side effects of chemotherapy such as nausea, for palliative purposes and to treat allergic reactions. While the potent pro-apoptotic properties and the supportive effects of GCs to tumour therapy in lymphoid cells are well studied, the impact of GCs to cytotoxic treatment of pancreatic carcinoma is unknown.

Methods

A prospective study of DEX-mediated resistance was performed using a pancreatic carcinoma xenografted to nude mice, 20 surgical resections and 10 established pancreatic carcinoma cell lines. Anti-apoptotic signaling in response to DEX was examined by Western blot analysis.

Results

In vitro, DEX inhibited drug-induced apoptosis and promoted the growth in all of 10 examined malignant cells. Ex vivo, DEX used in physiological concentrations significantly prevented the cytotoxic effect of gemcitabine and cisplatin in 18 of 20 freshly isolated cell lines from resected pancreatic tumours. No correlation with age, gender, histology, TNM and induction of therapy resistance by DEX co-treatment could be detected. In vivo, DEX totally prevented cytotoxicity of chemotherapy to pancreatic carcinoma cells xenografted to nude mice. Mechanistically, DEX upregulated pro-survival factors and anti-apoptotic genes in established pancreatic carcinoma cells.

Conclusion

These data show that DEX induces therapy resistance in pancreatic carcinoma cells and raise the question whether GC-mediated protection of tumour cells from cancer therapy may be dangerous for patients.     

Histone deacetylase inhibitors VPA and TSA induce apoptosis and autophagy in pancreatic cancer cells

Purpose

Histone deacetylase inhibitors (HDACi) are anti-neoplastic agents that are known to affect the growth of different cancer types, but their underlying mechanisms are still incompletely understood. Here, we compared the effects of two HDACi, i.e., Trichostatin A (TSA) and Valproic Acid (VPA), on the induction of cell death and autophagy in pancreatic cancer-derived cells that exhibit a high metastatic capacity and carry KRAS/p53 double mutations.

Methods

Cell viability and proliferation tests were carried out using Trypan blue dye exclusion, MTT and BrdU assays. FACS analyses were carried out to assess cell cycle progression, apoptosis, reactive oxygen species (ROS) production and mitochondrial depolarization, while Western blot and immunoprecipitation analyses were employed to detect proteins involved in apoptosis and autophagy.

Results

We found that both VPA and TSA can induce apoptosis in Panc1 and PaCa44 pancreatic cancer-derived cells by triggering mitochondrial membrane depolarization, Cytochrome c release and Caspase 3 activation, although VPA was more effective than TSA, especially in Panc1 cells. As underlying molecular events, we found that ERK1/2 was de-phosphorylated and that the c-Myc and mutant p53 protein levels were reduced after VPA and, to a lesser extent, after TSA treatment. Up-regulation of p21 and Puma was also observed, concomitantly with mutant p53 degradation. In addition, we found that in both cell lines VPA increased the pro-apoptotic Bim level, reduced the anti-apoptotic Mcl-1 level and increased ROS production and autophagy, while TSA was able to induce these effects only in PaCA44 cells.

Conclusions

From our results we conclude that both VPA and TSA can induce pancreatic cancer cell apoptosis and autophagy. VPA appears have a stronger and broader cytotoxic effect than TSA and, thus, may represent a better choice for anti-pancreatic cancer therapy.

     

Indole-3-carbinol synergistically sensitises ovarian cancer cells to bortezomib treatment

Background:

Bortezomib is a proteasome inhibitor with minimal clinical activity as a monotherapy in solid tumours, but its combination with other targeted therapies is being actively investigated as a way to increase its anticarcinogenic properties. Here, we evaluate the therapeutic potential of co-treatment with bortezomib and indole-3-carbinol (I3C), a natural compound found in cruciferous vegetables, in human ovarian cancer.

Methods:

We examined the effects of I3C, bortezomib and cisplatin in several human ovarian cancer cell lines. Synergy was determined using proliferation assays and isobologram analysis. Cell cycle and apoptotic effects were assessed by flow cytometry. The mechanism of I3C and bortezomib action was determined by RNA microarray studies, quantitative RT–PCR and western blotting. Antitumour activity of I3C and bortezomib was evaluated using an OVCAR5 xenograft mouse model.

Results:

I3C sensitised ovarian cancer cell lines to bortezomib treatment through potent synergistic mechanisms. Combination treatment with bortezomib and I3C led to profound cell cycle arrest and apoptosis as well as disruptions to multiple pathways, including those regulating endoplasmic reticulum stress, cytoskeleton, chemoresistance and carcinogen metabolism. Moreover, I3C and bortezomib co-treatment sensitised ovarian cancer cells to the standard chemotherapeutic agents, cisplatin and carboplatin. Importantly, in vivo studies demonstrated that co-treatment with I3C and bortezomib significantly inhibited tumour growth and reduced tumour weight compared with either drug alone.

Conclusion:

Together, these data provide a novel rationale for the clinical application of I3C and bortezomib in the treatment of ovarian cancer.