Arun Bhardwaj

CXCL12–CXCR4 signalling axis confers gemcitabine resistance to pancreatic cancer cells: a novel target for therapy

Background:Pancreatic cancer cells are highly resistant to drug therapy; however, underlying causes remain largely unknown. We hypothesised that the activation of CXCL12–CXCR4 signalling confers drug resistance to pancreatic cancer cells by potentiating survival. CXCR4 is overexpressed in precancerous/malignant pancreatic lesions and cancer stem cells, and implicated in its pathogenesis.Methods:Effect of CXCR4 activation by CXCL12 on restricting the gemcitabine-induced cytotoxicity and stimulating the survival signalling was examined in pancreatic cancer cells by MTT, DNA laddering, caspase activity, immunoblot, and promoter-reporter assays. Subsequently, we examined the effect of CXCR4 antagonist, AMD3100, in abrogating the rescue effect of activated CXCL12–CXCR4 signalling.Results:The pancreatic cancer cells treated with gemcitabine exhibited reduced cytotoxicity in the presence of CXCL12 as compared with the cells treated with drug alone. CXCL12 induced the activation of FAK, ERK, and Akt signalling pathways, enhanced transcriptional activities of β-catenin and NF-κB, and expression of survival proteins. AMD3100 arrested the CXCL12-induced pancreatic cancer cell growth and drug resistance.Conclusion:Our findings demonstrate, for the first time, a role of CXCL12–CXCR4 signalling axis in conferring drug resistance to pancreatic cancer cells and suggest that it could serve as a novel therapeutic target for pancreatic cancer therapy, alone and in combination with the cytotoxic drug.

Honokiol Arrests Cell Cycle, Induces Apoptosis, and Potentiates the Cytotoxic Effect of Gemcitabine in Human Pancreatic Cancer Cells

Survival rates for patients with pancreatic cancer are extremely poor due to its asymptomatic progression to advanced and metastatic stage for which current therapies remain largely ineffective. Therefore, novel therapeutic agents and treatment approaches are desired to improve the clinical outcome. In this study, we determined the effects of honokiol, a biologically active constituent of oriental medicinal herb Magnolia officinalis/grandiflora, on two pancreatic cancer cell lines, MiaPaCa and Panc1, alone and in combination with the standard chemotherapeutic drug, gemcitabine. Honokiol exerted growth inhibitory effects on both the pancreatic cancer cell lines by causing cell cycle arrest at G1 phase and induction of apoptosis. At the molecular level, honokiol markedly decreased the expression of cyclins (D1 and E) and cyclin-dependent kinases (Cdk2 and Cdk4), and caused an increase in Cdk inhibitors, p21 and p27. Furthermore, honokiol treatment led to augmentation of Bax/Bcl-2 and Bax/Bcl-xL ratios to favor apoptosis in pancreatic cancer cells. These changes were accompanied by enhanced cytoplasmic accumulation of NF-κB with a concomitant decrease in nuclear fraction and reduced transcriptional activity of NF-κB responsive promoter. This was associated with decreased phosphorylation of inhibitor of kappa B alpha (IκB-α) causing its stabilization and thus increased cellular levels. Importantly, honokiol also potentiated the cytotoxic effects of gemcitabine, in part, by restricting the gemcitabine-induced nuclear accumulation of NF-κB in the treated pancreatic cancer cell lines. Altogether, these findings demonstrate, for the first time, the growth inhibitory effects of honokiol in pancreatic cancer and indicate its potential usefulness as a novel natural agent in prevention and therapy.

CXCL12/CXCR4 Protein Signaling Axis Induces Sonic Hedgehog Expression in Pancreatic Cancer Cells via Extracellular Regulated Kinase- and Akt Kinase-mediated Activation of Nuclear Factor κB IMPLICATIONS FOR BIDIRECTIONAL TUMOR-STROMAL INTERACTIONS

Background: CXCL12/CXCR4 and hedgehog pathways, predominantly acting in paracrine fashion, play important roles in pancreatic cancer pathobiology. Results: CXCL12/CXCR4 signaling regulates the expression of hedgehog ligand, the sonic hedgehog, in pancreatic cancer cells. Conclusion: Our findings indicate a novel molecular link between CXCL12/CXCR4 and hedgehog pathways. Significance: Our data provide a molecular basis for an active bidirectional tumor-stromal interaction in pancreatic cancer. Recent evidence suggests a major role of tumor-stromal interactions in pancreatic cancer pathobiology. The chemokine CXCL12 (stromal cell-derived factor 1 (SDF-1)), abundantly produced by stromal cells, promotes progression, metastasis, and chemoresistance of pancreatic cancer cells. On the other hand, pancreatic tumor cell-derived sonic hedgehog (SHH) acts predominantly on stromal cells to induce desmoplasia and, thus, has a paracrine effect on tumorigenesis and therapeutic outcome. In this study, we examined the association between these two proteins of pathological significance in pancreatic cancer. Our data demonstrate that CXCL12 leads to a dose- and time-dependent up-regulation of SHH in pancreatic cancer cells. CXCL12-induced SHH up-regulation is specifically mediated through the receptor CXCR4 and is dependent on the activation of downstream Akt and ERK signaling pathways. Both Akt and ERK cooperatively promote nuclear accumulation of NF-κB by inducing the phosphorylation and destabilization of its inhibitory protein, IκB-α. Using dominant negative IκB-α, a SHH promoter (deletion mutant) reporter, and chromatin immunoprecipitation assays, we demonstrate that CXCL12 exposure enhances direct binding of NF-κB to the SHH promoter and that suppression of NF-κB activation abrogates CXCL12-induced SHH expression. Finally, our data demonstrate a strong correlative expression of CXCR4 and SHH in human pancreatic cancer tissues, whereas their expression is not observed in the normal pancreas. Altogether, our data reveal a novel mechanism underlying aberrant SHH expression in pancreatic cancer and identify a molecular link facilitating bidirectional tumor-stromal interactions.

An Undesired Effect of Chemotherapy GEMCITABINE PROMOTES PANCREATIC CANCER CELL INVASIVENESS THROUGH REACTIVE OXYGEN SPECIES-DEPENDENT, NUCLEAR FACTOR κB- AND HYPOXIA-INDUCIBLE FACTOR 1α-MEDIATED UP-REGULATION OF CXCR4

Background: CXCR4 signaling protects pancreatic cancer cells from gemcitabine toxicity. However, the effect of gemcitabine on this resistance mechanism is unclear. Results: Gemcitabine up-regulates CXCR4 expression in pancreatic cancer cells and promotes their invasiveness. Conclusion: CXCR4 signaling serves as a counterdefense mechanism against gemcitabine. Significance: These findings are significant for the formulation of effective therapeutic strategies against pancreatic cancer. Recently, we have shown that CXCL12/CXCR4 signaling plays an important role in gemcitabine resistance of pancreatic cancer (PC) cells. Here, we explored the effect of gemcitabine on this resistance mechanism. Our data demonstrate that gemcitabine induces CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time-dependent manner. Gemcitabine-induced CXCR4 expression is dependent on reactive oxygen species (ROS) generation because it is abrogated by pretreatment of PC cells with the free radical scavenger N-acetyl-L-cysteine. CXCR4 up-regulation by gemcitabine correlates with time-dependent accumulation of NF-κB and HIF-1α in the nucleus. Enhanced binding of NF-κB and HIF-1α to the CXCR4 promoter is observed in gemcitabine-treated PC cells, whereas their silencing by RNA interference causes suppression of gemcitabine-induced CXCR4 expression. ROS induction upon gemcitabine treatment precedes the nuclear accumulation of NF-κB and HIF-1α, and suppression of ROS diminishes these effects. The effect of ROS on NF-κB and HIF-1α is mediated through activation of ERK1/2 and Akt, and their pharmacological inhibition also suppresses gemcitabine-induced CXCR4 up-regulation. Interestingly, our data demonstrate that nuclear accumulation of NF-κB results from phosphorylation-induced degradation of IκBα, whereas HIF-1α up-regulation is NF-κB-dependent. Lastly, our data demonstrate that gemcitabine-treated PC cells are more motile and exhibit significantly greater invasiveness against a CXCL12 gradient. Together, these findings reinforce the role of CXCL12/CXCR4 signaling in gemcitabine resistance and point toward an unintended and undesired effect of chemotherapy.

Modulation of Protein Phosphatase 2A Activity Alters Androgen-Independent Growth of Prostate Cancer Cells: Therapeutic Implications

Earlier we identified PPP2CA, which encodes for the α-isoform of protein phosphatase 2A (PP2A) catalytic subunit, as one of the downregulated genes in androgen-independent prostate cancer. PP2A is a serine/threonine phosphatase and a potent tumor suppressor involved in broad cellular functions; however, its role in prostate cancer has not yet been determined. Here, we have investigated the effect of PP2A activity modulation on the androgen-independent growth of prostate cancer cells. Our data show that the PPP2CA expression and PP2A activity is downregulated in androgen-independent (C4-2) prostate cancer cells as compared with androgen-dependent (LNCaP) cells. Downregulation of PP2A activity by pharmacologic inhibition or short interfering RNA-mediated PPP2CA silencing sustains the growth of LNCaP cells under an androgen-deprived condition by relieving the androgen deprivation–induced cell-cycle arrest and preventing apoptosis. Immunoblot analyses reveal enhanced phosphorylation of Akt, extracellular signal–regulated kinase (ERK), BAD, increased expression of cyclins (A1/D1), and decreased expression of cyclin inhibitor (p27) on PP2A downregulation. Furthermore, our data show that androgen receptor (AR) signaling is partially maintained in PP2A-inhibited cells through increased AR expression and ligand-independent phosphorylation. Pharmacologic inhibition of Akt, ERK, and AR suggest a role of these signaling pathways in facilitating the androgen-independent growth of LNCaP cells. These observations are supported by the effect of ceramide, a PP2A activator, on androgen-independent C4-2 cells. Ceramide inhibited the growth of C4-2 cells on androgen deprivation, an effect that could be abrogated by PP2A downregulation. Altogether, our findings suggest that modulation of PP2A activity may represent an alternative therapeutic approach for the treatment of advanced androgen-independent prostate cancer. Mol Cancer Ther; 10(5); 720–31. ©2011 AACR.

Silver nanoparticles protect human keratinocytes against UVB radiation-induced DNA damage and apoptosis: potential for prevention of skin carcinogenesis.

UNLABELLED Ultraviolet (UV)-B radiation from the sun is an established etiological cause of skin cancer, which afflicts more than a million lives each year in the United States alone. Here, we tested the chemopreventive efficacy of silver-nanoparticles (AgNPs) against UVB-irradiation-induced DNA damage and apoptosis in human immortalized keratinocytes (HaCaT). AgNPs were synthesized by reduction-chemistry and characterized for their physicochemical properties. AgNPs were well tolerated by HaCaT cells and their pretreatment protected them from UVB-irradiation-induced apoptosis along with significant reduction in cyclobutane-pyrimidine-dimer formation. Moreover, AgNPs pre-treatment led to G1-phase cell-cycle arrest in UVB-irradiated HaCaT cells. AgNPs were efficiently internalized in UVB-irradiated cells and localized into cytoplasmic and nuclear compartments. Furthermore, we observed an altered expression of various genes involved in cell-cycle, apoptosis and nucleotide-excision repair in HaCaT cells treated with AgNPs prior to UVB-irradiation. Together, these findings provide support for potential utility of AgNPs as novel chemopreventive agents against UVB-irradiation-induced skin carcinogenesis. FROM THE CLINICAL EDITOR Excessive exposure to the sun is known to increase the risk of skin cancer due to DNA damage. In this work, the authors tested the use of silver nanoparticles as protective agents against ultraviolet radiation. The positive results may open a door for the use of silver nanoparticle as novel agents in the future.

Myb overexpression overrides androgen depletion–induced cell cycle arrest and apoptosis in prostate cancer cells, and confers aggressive malignant traits: potential role in castration resistance

Myb, a cellular progenitor of v-Myb oncogenes, is amplified in prostate cancer and exhibits greater amplification frequency in hormone-refractory disease. Here, we have investigated the functional significance of Myb in prostate cancer. Our studies demonstrate Myb expression in all prostate cancer cell lines (LNCaP, C4-2, PC3 and DU145) examined, whereas it is negligibly expressed in normal/benign prostate epithelial cells (RWPE1 and RWPE2). Notably, Myb is significantly upregulated, both at transcript (>60-fold) and protein (>15-fold) levels, in castration-resistant (C4-2) cells as compared with androgen-dependent (LNCaP) prostate cancer cells of the same genotypic lineage. Using loss and gain of function approaches, we demonstrate that Myb promotes and sustains cell cycle progression and survival under androgen-supplemented and -deprived conditions, respectively, through induction of cyclins (A1, D1 and E1), Bcl-xL and Bcl2 and downregulation of p27 and Bax. Interestingly, Myb overexpression is also associated with enhanced prostate-specific antigen expression. Furthermore, our data show a role of Myb in enhanced motility and invasion and decreased homotypic interactions of prostate cancer cells. Myb overexpression is also associated with actin reorganization leading to the formation of filopodia-like cellular protrusions. Immunoblot analyses demonstrate gain of mesenchymal and loss of epithelial markers and vice versa, in Myb-overexpressing LNCaP and -silenced C4-2 cells, respectively, indicating a role of Myb in epithelial to mesenchymal transition. Altogether, our studies provide first experimental evidence for a functional role of Myb in growth and malignant behavior of prostate cancer cells and suggest a novel mechanism for castration resistance.

Restoration of PPP2CA expression reverses epithelial-to-mesenchymal transition and suppresses prostate tumour growth and metastasis in an orthotopic mouse model

Background:Emergence of castration-resistance in prostate cancer (PCa) is invariably associated with aggressive and metastatic disease. Previously, we reported promotion of castration-resistance upon downregulation of PPP2CA (encoding catalytic subunit of protein phosphatase 2A (PP2A), α-isoform); however, its role in PCa growth and metastasis remained undetermined.Methods:PPP2CA was overexpressed/silenced in PCa cells by stable transfection. Gene expression was examined by reverse transcription polymerase chain reaction, immunoblot and immunofluorescence analyses, and transcriptional activity measured by luciferase-based promoter-reporter assay. Effect on PCa phenotype was studied in vitro and in orthotopic mouse model, and immunohistochemical/histological analyses performed to assess proliferation/apoptosis and confirm metastatic lesions.Results:An inverse association of PPP2CA expression was observed with epithelial-to-mesenchymal transition (EMT) and aggressive PCa phenotype. PPP2CA restoration resulted in decreased nuclear accumulation and transcriptional activity of β-catenin/NF-κB, and restitution of their activity abrogated PPP2CA-induced EMT reversal and suppression of PCa invasiveness. Akt mediated PPP2CA loss-induced nuclear accumulation of β-catenin/NF-κB through inactivation of Gsk3-β and IκB-α, respectively. Animal studies revealed a suppressive effect of PPP2CA expression on PCa growth and metastasis.Conclusions:Our findings suggest that PPP2CA downregulation serves as a molecular link between gain of castration-resistance and aggressive PCa phenotype, and its restoration could be an effective preventive/therapeutic approach against the advanced disease.

p-21 activated kinase 4 (PAK4) maintains stem cell-like phenotypes in pancreatic cancer cells through activation of STAT3 signaling

Pancreatic cancer (PC) remains a highly lethal malignancy due to its unusual chemoresistance and high aggressiveness. A subpopulation of pancreatic tumor cells, known as cancer stem cells (CSCs), is considered responsible not only for tumor-maintenance, but also for its widespread metastasis and therapeutic failure. Here we investigated the role of p-21 activated kinase 4 (PAK4) in driving PC stemness properties. Our data demonstrate that triple-positive (CD24+/CD44+/EpCAM+) subpopulation of pancreatic CSCs exhibits greater level of PAK4 as compared to triple-negative (CD24−/CD44−/EpCAM−) cells. Moreover, PAK4 silencing in PC cells leads to diminished fraction of CD24, CD44, and EpCAM positive cells. Furthermore, we show that PAK4-silenced PC cells exhibit decreased sphere-forming ability and increased chemo-sensitivity to gemcitabine toxicity. PAK4 expression is also associated with enhanced levels of stemness-associated transcription factors (Oct4/Nanog/Sox2 and KLF4). Furthermore, our data show decreased nuclear accumulation and transcriptional activity of STAT3 in PAK4-silenced PC cells and restitution of its activity leads to restoration of stem cell phenotypes. Together, our findings deliver first experimental evidence for the involvement of PAK4 in PC stemness and support its clinical utility as a novel therapeutic target in PC.

Synthesis, characterization, and evaluation of poly (D,L-lactide-co-glycolide)-based nanoformulation of miRNA-150: potential implications for pancreatic cancer therapy.

MicroRNAs are small (18–22 nucleotide long) noncoding RNAs that play important roles in biological processes through posttranscriptional regulation of gene expression. Their aberrant expression and functional significance are reported in several human malignancies, including pancreatic cancer. Recently, we identified miR-150 as a novel tumor suppressor microRNA in pancreatic cancer. Furthermore, expression of miR-150 was downregulated in the majority of tumor cases, suggesting that its restoration could serve as an effective approach for pancreatic cancer therapy. In the present study, we developed a nanoparticle-based miR-150 delivery system and tested its therapeutic efficacy in vitro. Using double emulsion solvent evaporation method, we developed a poly (D,L-lactide-co-glycolide) (PLGA)-based nanoformulation of miR-150 (miR-150-NF). Polyethyleneimine (a cationic polymer) was incorporated in PLGA matrix to increase the encapsulation of miR-150. Physical characterization of miR-150-NF demonstrated that these nanoparticles had high encapsulation efficiency (~78%) and exhibited sustained release profile. Treatment of pancreatic cancer cells with miR-150-NF led to efficient intracellular delivery of miR-150 mimics and caused significant downregulation of its target gene (MUC4) expression. Inhibition of MUC4 correlated with a concomitant decrease in the expression of its interacting partner, HER2, and repression of its downstream signaling. Furthermore, treatment of pancreatic cancer cells with miR-150-NF suppressed their growth, clonogenicity, motility, and invasion. Together, these findings suggest that PLGA-based nanoformulation could potentially serve as a safe and effective nanovector platform for miR-150 delivery to pancreatic tumor cells.