One of the major challenges associated with cancer therapy is the acquisition of chemoresistance by tumour cells. Many novel therapeutic approaches to overcome chemoresistance have involved targeting specific signalling pathways such as the phosphatidylinositol 3-kinase (PI3K) pathway, a stress response pathway known to be involved in the regulation of cell survival, apoptosis and growth. The present study sought to determine the effect of PI3K inhibition on the resistance of human cancer cells to various chemotherapeutic agents. Treatment with the PI3K inhibitors LY294002 or Compound 15e resulted in resistance to doxorubicin, etoposide, 5-fluorouracil, and vincristine in breast (MDA-MB-231) carcinoma cells as determined by clonogenic assays. Increased survival following PI3K inhibition was also observed in prostate (DU-145), colon (HCT-116) and lung (A-549) carcinoma cell lines exposed to doxorubicin. Drug resistance mediated by LY294002 was correlated with a decrease in cell proliferation, which was linked to an increase in the proportion of cells in the G1 phase of the cell cycle. Inhibition of PI3K signalling also resulted in higher levels of the cyclin-dependent kinase inhibitors p21Waf1/Cip1 and p27Kip1. Knockdown of p21Waf1/Cip1 expression with siRNA resulted in a significant decrease in LY294002-induced resistance. However, the effect of p27Kip1 knockdown on LY294002-induced resistance was inconclusive because of high inter-experimental variability. Furthermore, knockdown of either p21waf1/cip1 or p27Kip1 did not overtly prevent LY294002-induced cell cycle delay. Incubation in the presence of LY294002 after exposure to doxorubicin resulted in decreased cell survival. These findings provide evidence that PI3K inhibition leads to chemoresistance in human cancer cells by causing a delay in cell cycle. However, the timing of PI3K inhibition (either before or after exposure to anti-cancer agents) is a critical determinant of chemosensitivity. Since the efficiency of most chemotherapeutic agents depends on the rate of cell proliferation, delays in cell cycle progression may be an important mechanism of chemoresistance. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2008-09-24 14:00:39.679

A novel gene, named as the PHDX gene, had been previously identified while screening genes for their involvement in resistance to the chemotherapeutic drug etoposide and to hydrogen peroxide, using the methodology of retrovirus promoter trap mutagenesis. This study was undertaken for the purpose of testing whether the loss of PHDX gene is responsible for drug resistance in CHO-E-126 cell line which was created from Chinese hamster ovary cells having a retroviral receptor and selected for etoposide resistance. The PHDX gene resides on mouse chromosome 11 and has homology with the prolyl hydroxylase gene family. We hypothesized that the inactivation of the PHDX gene by promoter trap mutagenesis will confer resistance to etoposide and hydrogen peroxide in the E-126 cell line. In addition, the alteration of the cellular hydroxyproline levels by the loss of the gene might influence the drug response through the production of oxygen free radicals. To study the involvement of the PHDX gene in etoposide and hydrogen peroxide induced drug-resistance, we used two experimental approaches to modulate the function of the gene in cells. First, we silenced the expression of this gene by RNA interference (RNAi) through stable and transient knockdown experiments in the parental Chinese hamster ovary (CHO-K1)cells, and second, we overexpressed the gene in CHO-Cl-22 and CHO-E-126 cells. We assessed the effect of the knockdown by RT-PCR. The effect of etoposide and hydrogen peroxide was determined by the clonogenic crystal violet staining assay and the MTT assay. Through our siRNAi knockdown studies, we were able to demonstrate the involvement of the gene in drug resistance. We were unable to show that the overexpression of the gene was capable of reverting to the drug sensitive phenotype.

A novel gene, named as the PHDX gene, had been previously identified while screening genes for their involvement in resistance to the chemotherapeutic drug etoposide and to hydrogen peroxide, using the methodology of retrovirus promoter trap mutagenesis. This study was undertaken for the purpose of testing whether the loss of PHDX gene is responsible for drug resistance in CHO-E-126 cell line which was created from Chinese hamster ovary cells having a retroviral receptor and selected for etoposide resistance. The PHDX gene resides on mouse chromosome 11 and has homology with the prolyl hydroxylase gene family. We hypothesized that the inactivation of the PHDX gene by promoter trap mutagenesis will confer resistance to etoposide and hydrogen peroxide in the E-126 cell line. In addition, the alteration of the cellular hydroxyproline levels by the loss of the gene might influence the drug response through the production of oxygen free radicals. To study the involvement of the PHDX gene in etoposide and hydrogen peroxide induced drug-resistance, we used two experimental approaches to modulate the function of the gene in cells. First, we silenced the expression of this gene by RNA interference (RNAi) through stable and transient knockdown experiments in the parental Chinese hamster ovary (CHO-K1)cells, and second, we overexpressed the gene in CHO-Cl-22 and CHO-E-126 cells. We assessed the effect of the knockdown by RT-PCR. The effect of etoposide and hydrogen peroxide was determined by the clonogenic crystal violet staining assay and the MTT assay. Through our siRNAi knockdown studies, we were able to demonstrate the involvement of the gene in drug resistance. We were unable to show that the overexpression of the gene was capable of reverting to the drug sensitive phenotype.

No description available.

<div>Cancers have the ability to develop resistance to traditional therapies. The important role of the tumor microenvironment in transforming nonaggressive tumor cells into an aggressive and chemoresistant cancer has been abundantly addressed. Mechanical cues from the tumor environment, such as matrix stiffness and geometry, transfer to the cell nucleus via the cytoskeleton and change nuclear morphology (e. g, chromatin organization, size and shape). Such alterations are known to accompany or follow the acquisition of chemoresistance. Nuclear matrix proteins such as the Nuclear Mitotic Apparatus (NuMA) are highly involved in higher order chromatin organization and contribute to sustain the physical structure of the cell nucleus, but it is</div><div>yet to be determined how such structural proteins respond to microenvironmental changes. We have shown previously that tumors cultured in curved geometry (similar to the ductal architecture of breast tissue) display significantly different drug sensitivities compared to those cultured on a flat surface, and that a major morphological difference between these two culture conditions is nuclear shape (i.e., circularity). Our hypothesis is that mechanical cues from the tumor microenvironment alter nuclear features that control the phenotypic response of cancer cells to antiproliferative drugs. Morphological analysis of the cell nucleus in the curved conformation as well as hydrogel and hanging drop systems (with amorphous geometry) showed that only nodules in the curved set-up have nuclear morphometry (shape and size) similar to that of breast tumors of the corresponding subtypes in vivo. In addition, we compared the sensitivity of triple negative breast tumors to cisplatin, with proven efficacy in the clinics, and SAHA, an epigenetic drug that so far failed in breast cancer treatment. Our results suggest higher sensitivity to cisplatin and lower sensitivity to SAHA of breast cancer cells cultured in duct-like geometry compared to the amorphous systems. To evaluate the importance of nuclear morphometry in drug response we altered nuclear size and shape using latrunculin. Under this condition, the number of apoptotic and growth-arrested cells increased following treatments with cisplatin and SAHA, respectively.</div>

No description available.

First-line treatment of breast and other cancers with Taxol is compromised by resistance in up to 40% of patients. To improve chemotherapy, it is vital to understand how Taxol resistance develops and to overcome this. Autotaxin (ATX) promotes cancer cell survival, growth, migration, invasion and metastasis. ATX converts extracellular lysophosphatidylcholine (LPC) into lysophosphatidate (LPA). As these lipids have been reported to affect cell signaling through their own G-protein-coupled receptors, ATX could modify the balance of this signaling. Also, ATX affects cell adhesion independently of its catalytic activity, We first investigated the interactions of ATX, LPC and LPA on the apoptotic effects of Taxol, which is commonly used in breast cancer treatment. LPC had no significant effect on Taxol-induced apoptosis in MCF-7 breast cancer cells, which do not secrete significant ATX. Addition of incubation medium from MDA-MB-435 melanoma cells, which secrete ATX, or recombinant ATX enabled LPC to inhibit Taxol-induced apoptosis of MCF-7 cells. Inhibiting ATX activity blocked this protection against apoptosis. We conclude that LPC has no significant effect in protecting MCF-7 cells against Taxol treatment unless it is converted to LPA by ATX. LPA strongly antagonized Taxol-induced apoptosis through stimulating phosphatidylinositol 3-kinase and inhibiting ceramide formation. LPA also partially reversed the Taxol-induced arrest in the G2/M phase of the cell cycle. Then, we described a novel action of LPA, which by activating phosphatidylinositol 3-kinase increases the expression of glycogen synthase kinase-3 and survivin. Survivin is an anti-apoptotic protein, which also increases the dynamicity of microtubules. Survivin decreased the effectiveness of Taxol in stabilizing microtubules and enabled MCF-7 breast cancer cells to escape from Taxol-induced arrest in G2/M and consequent cell death. Our work showed that inhibiting ATX activity and LPA-mediated signaling can reverse the resistance to Taxol-induced cell death. Our results support the hypothesis that therapeutic inhibition of ATX activity, which results in less LPA production, or inhibition of LPA signalling could improve the efficacy of Taxol as a chemotherapeutic agent for cancer treatment. / Medical Sciences - Laboratory Medicine and Pathology

Cisplatin (CDDP; cis-diamminedichloroplatinum) resistance presents a major impediment in the treatment of several gynecologic solid tumors, including ovarian and cervical tumors. p53, a critical regulator of cellular apoptosis, is a determinant of CDDP sensitivity. In our study, we have observed that the dysregulation of p53 regulators, checkpoint kinase 1 (Chk1) and protein phosphatase magnesium-dependent 1 (PPM1D), significantly reduced CDDP responsiveness in human cancer cells. Isogenic wt-p53 CDDP-sensitive (OV2008) and -resistant (C13*) cervical cancer cells, and isogenic wt-p53 CDDP-sensitive (A2780s) and p53 mutant resistant (A2780cp) ovarian cancer cells, along with CDDP-resistant ovarian cancer cell lines (OCC-1 and OVCAR-3, mutant p53; SKOV-3, p53 null) were used to elucidate the mechanisms of p53 regulation in human gynecologic cancer cells. We have complemented our study with a xenograft model (A2780s) and a tissue microarray of human ovarian tumors to validate our in vitro observations. We have demonstrated that CDDP differentially regulated the p53 activator Chk1 in sensitive and resistant cancer cells; it enhances Chk1 activation in sensitive but not resistant cells. This differential regulation also extended to PPM1D, whereby CDDP enhanced PPM1D content in resistant but not sensitive cells. PPM1D knockdown sensitized resistant cells to CDDP, which was associated with up-regulation of Chk1 and p53 activations, while PPM1D over-expression had the opposite effect. We have also shown that CDDP sensitivity in response to PPM1D down-regulation was p53-dependent. Moreover, CDDP promotes PPM1D nuclear localization in resistant cells and nuclear exclusion in sensitive cells and xenograft tumors. Enhanced PPM1D expression in human ovarian tumors is significantly associated with tumor aggression. Dysregulation of the oncogene Akt has been implicated in a variety of human malignancies, including ovarian cancer. We have demonstrated that Akt regulates PPM1D stability, since activated Akt over-expression in sensitive cells rescued PPM1D from CDDP-induced proteasomal degradation and Akt down-regulation in resistant cells lead to PPM1D de-stabilization and down-regulation. We have shown for the first time that PPM1D is downstream of Akt through which it can modulate CDDP sensitivity in human cancer cells. These findings extend the current knowledge on the molecular basis of CDDP resistance in gynecological cancers and may help in developing effective therapeutic strategies.

Mammalian homolog Tribbles (Trbs) is a newly characterized protein family that includes three different isoforms: Trb1, Trb2, and Trb3. Tribbles are serine/threonine kinases lacking catalytic activity, thus their classification as pseudokinases. Despite their catalytic inactivity, Tribbles can interact with different proteins and regulate different biological functions. The most studied tribble family member, Trb3, was reported to play a major role in Drosophila's ventral furrow formation. Further studies revealed that Trb3 is also involved in diabetes, stress-response, and development. Previously, Trb3 upregulation was detected in certain types of cancer but its function remains unknown. The goal of our study is to gain a better understanding of the biological function of Trb3 in cancer, including the molecular mechanism of action. Using the cohort analysis, we identified higher levels of Trb3 in the lung tumor compared to the normal tissue. Furthermore, higher Trb3 expression in the lung tissue was associated with a poor survival in cancer patients. Silencing of Trb3 in A549 promoted cell growth. On the other hand, overexpression of Trb3 in NCI-H358 inhibited cell growth. The analysis of cell cycle gene profiling revealed a decrease in several genes that are essential for cell cycle progression in S phase in Trb3 overexpressed NCI-H358. The cell proliferation protein, Ki67, was also decreased in Trb3 overexpressed NCI-H358 cells. Moreover, Tb3 overexpressed cells formed higher colony number in soft agar assay and depicted higher migration ability in the Boyden chamber assay. Mesenchymal markers SNAIL, TWIST and N-cadherin were upregulated while epithelial E-cadherin was significantly reduced. Interestingly, prosurvival protein Akt was also reduced post Trb3 overexpression. Trb3 expression was associated with a poor survival. However, we discovered that Trb3 overexpression inhibited cell growth. Thus, we hypothesized that Trb3 expression might contribute to tumorigenesis during cellular metabolic stress. In order to understand the potential role of Trb3 in metabolic stress, NCI-H358 cells were treated with five different cellular stressors to mimic the tumor microenvironment. All stressors used were shown to induce endogenous Trb3 expression. Moreover, stress proteins ATF4, CHOP and ASNS were induced by all stressors. One of the stressors used was rotenone, an inhibitor of the complex I of the electron transport chain. Rotenone treatment induced Trb3 expression. This expression inversely correlated with cytochrome C expression. Furthermore, Trb3 expression positively correlated with the expression of mitophagic genes PINK1, Parkin and p62, which suggest that Trb3 is induced during ROS-mediated oxidative stress to participate in the clearing of damaged mitochondria. This targeted clearing of the mitochondria, a process known as mitophagy is essential for the cell survival of the lung cancer cells. Last, Trb3 overexpression rendered cancer cells resistant to docetaxel and cisplatin, two chemotherapeutic drugs used in lung cancer treatment. On the other hand, Trb3 depleted cells were more sensitive to the drugs. Our results suggest that Trb3 is activated in the primary tumor to promote metabolic adaptation through cell cycle arrest and the inhibition of aerobic glucose metabolism through Akt inhibition. Furthermore, Trb3 is essential during cell survival post ROS-mediated stress and participates in the clearing of damaged mitochondria during mitophagy. Last, stress-mediated activation of Trb3 confers lung cancer cells with chemoresistance and suggest that Trb3 could be a potential target in lung cancer therapy.

OVCA is the most lethal gynecological cancer, due primarily to late diagnosis and chemoresistance (Canada, 2014; Society, 2014b). CDDP resistance is a major hurdle to successful therapy (MayoClinic, 2014). The mechanism of chemoresistance is multi-factorial including defects in apoptotic pathway and key tumor suppressor as well as dysregulation of metabolism (Borst et al., 2000; Galluzzi et al., 2012a; Siddik, 2003). Elevated aerobic glycolysis is a major source for fulfilling high energy demand of cancer, but the role of metabolic reprogramming and its regulatory mechanism in OVCA cells remain unknown. p53 is a key tumor suppressor involved in apoptosis and frequent defect of p53 (> 80%) exist in epithelial OVCA. HKII is a key metabolic enzyme involved in the first step of glycolysis and its frequent presence in the mitochondria (80% >) has been reported in multiple cancers. We demonstrate here that CDDP-induced, p53-mediated HKII down-regulation and mitochondrial p53-HKII interaction are determinants of chemosensitivity in OVCA. CDDP decreased HKII (mRNA abundance, protein level), altered its cellular localization and glycolysis in p53-wt chemosensitive OVCA cells, a response loss or attenuated in p53 deficient cells. HKII depletion sensitized chemoresistant cells to CDDP -induced apoptosis in a p53- dependent manner. In addition, p53 binds to HKII and facilitates its nuclear localization. Mechanistically, our data suggest that CDDP-activated p53 (phosphorylated p53; P-p53 Ser15) interacts with HKII in the nucleus for its regulation. Upon entry to the nucleus, P-p53(Ser15) transcriptionally regulates HKII by promoter binding, contributing to the regulation of HKII and aerobic glycolysis, eliciting apoptosis in chemosensitive OVCA cells. Conversely, this response is compromised in p53 defect chemoresistant cells. Using proximity ligation assay (PLA) in human OVCA cell lines and primary tumor cells and tumor sections from OVCA patients, we have demonstrated that nuclear HKII-P-p53(Ser15) intracellular trafficking is associated with chemosensitivity in vitro and in vivo. Furthermore, the nuclear HKII-P-p53(Ser15) interaction may be useful as a biomarker for chemosensitivity in multiple epithelial subtypes of OVCA.