Similarities and Differences of Hsp70, hsc70, Grp78 and Mortalin as Cancer Biomarkers and Drug Targets.

Background: Upregulation of Heath Shock Protein 70 (HSP70) chaperones supports cancer cell survival. Their high homology causes a challenge to differentiate them in experimental or prevention and treatment strategies. The objective of this investigation was to determine similarities and differences of Hsp70, hsc70, Grp78 and Mortalin members of the HSP70 family encoded by HSPA1, HSPA8, HSPA5 and HSPA9 genes, respectively. Methods: Literature reviews were conducted using HSPA1, HSPA5, HSPA8 and HSPA9 gene or protein names or synonyms combined with biological or cancer-relevant terms. Ingenuity Pathway Analysis was used to identify and compare profiles of proteins that directly bind individual chaperones and their associated pathways. TCGA data was probed to identify associations of hsc70 with cancer patient survival. ClinicalTrials.gov was used to identify HSP70 family studies. Results: The chaperones have similar protein folding functions. Their different cellular effects are determined by co-chaperones and client proteins combined with their intra- and extra-cellular localizations. Their upregulation is associated with worse patient prognosis in multiple cancers and can stimulate tumor immune responses or drug resistance. Their inhibition selectively kills cancer over healthy cells. Conclusions: Differences in Hsp70, hsc70, Grp78 and mortalin provide opportunities to calibrate HSP70 inhibitors for individual cancers and combination therapies.

Potential and mechanism of mebendazole for treatment and maintenance of ovarian cancer.

OBJECTIVE: Mebendazole and other anti-parasitic drugs are being used off-prescription based on social media and unofficial accounts of their anti-cancer activity. The purpose of this study was to conduct a controlled evaluation of mebendazole's therapeutic efficacy in cell culture and in vivo models of ovarian cancer. The majority of ovarian cancers harbor p53 null or missense mutations, therefore the effects of p53 mutations and a mutant p53 reactivator, PRIMA-1MET (APR246) on mebendazole activity were evaluated. METHODS: Mebendazole was evaluated in cisplatin-resistant high grade serous stage 3C ovarian cancer patient derived xenograft (PDX) models: PDX-0003 (p53 null) and PDX-0030 (p53 positive), and on ovarian cancer cell lines: MES-OV (p53 R282W), ES2 (p53 S241F), A2780 (p53 wild type), SKOV3 parental (p53 null) and isogenic sublines, SKOV3 R273H p53 and SKOV3 R248W p53. Drug synergy and mechanisms were evaluated in cell cultures using isobolograms, clonogenic assays and western blots. Prevention of tumor establishment was studied in a MES-OV orthotopic model. RESULTS: Mebendazole inhibited growth of ovarian cancer cell cultures at nanomolar concentrations and PDXs at doses up to 50 mg/kg, and reduced orthotopic tumor establishment at 50 mg/kg. The mechanism of mebendazole was associated with p53-independent induction of p21 and tubule depolymerization. PRIMA-1MET also inhibited tumor establishment and worked synergistically with mebendazole in cell culture to inhibit growth and induce intrinsic apoptosis through a p53- and tubule destabilization-independent mechanism. CONCLUSION: This work demonstrates the therapeutic potential of repurposing mebendazole and supports clinical development of mebendazole for ovarian cancer therapy and maintenance.

Complementary Targeting of Rb Phosphorylation and Growth in Cervical Cancer Cell Cultures and a Xenograft Mouse Model by SHetA2 and Palbociclib.

Cervical cancer is caused by high-risk human papillomavirus (HPV) types and treated with conventional chemotherapy with surgery and/or radiation. HPV E6 and E7 proteins increase phosphorylation of retinoblastoma (Rb) by cyclin D1/cyclin dependent kinase (CDK)4/6 complexes. We hypothesized that cyclin D1 degradation by the SHetA2 drug in combination with palbociclib inhibition of CDK4/6 activity synergistically reduces phosphorylated Rb (phospho-Rb) and inhibits cervical cancer growth. The effects of these drugs, alone, and in combination, were evaluated in SiHa and CaSki HPV-positive and C33A HPV-negative cervical cancer cell lines using cell culture, western blots and ELISA, and in a SiHa xenograft model. Endpoints were compared by isobolograms, ANOVA, and Chi-Square. In all cell lines, combination indexes documented synergistic interaction of SHetA2 and palbociclib in association SHetA2 reduction of cyclin D1 and phospho-Rb, palbociclib reduction of phospho-Rb, and enhanced phospho-Rb reduction upon drug combination. Both drugs significantly reduced phospho-Rb and growth of SiHa xenograft tumors as single agents and acted additively when combined, with no evidence of toxicity. Dilated CD31-negative blood vessels adjacent to, or within, areas of necrosis and apoptosis were observed in all drug-treated tumors. These results justify development of the SHetA2 and palbociclib combination for targeting phospho-Rb in cervical cancer treatment.

Novel ovarian cancer maintenance therapy targeted at mortalin and mutant p53.

Current ovarian cancer maintenance therapy is limited by toxicity and no proven impact on overall survival. To study a maintenance strategy targeted at missense mutant p53, we hypothesized that the release of mutant p53 from mortalin inhibition by the SHetA2 drug combined with reactivation of mutant p53 with the PRIMA-1MET drug inhibits growth and tumor establishment synergistically in a mutant-p53 dependent manner. The Cancer Genome Atlas (TCGA) data and serous ovarian tumors were evaluated for TP53 and HSPA9/mortalin status. SHetA2 and PRIMA-1MET were tested in ovarian cancer cell lines and fallopian tube secretory epithelial cells using isobolograms, fluorescent cytometry, Western blots and ELISAs. Drugs were administered to mice after peritoneal injection of MESOV mutant p53 ovarian cancer cells and prior to tumor establishment, which was evaluated by logistic regression. Fifty-eight percent of TP53 mutations were missense and there were no mortalin mutations in TCGA high-grade serous ovarian cancers. Mortalin levels were sequentially increased in serous benign, borderline and carcinoma tumors. SHetA2 caused p53 nuclear and mitochondrial accumulation in cancer, but not in healthy, cells. Endogenous or exogenous mutant p53 increased SHetA2 resistance. PRIMA-1MET decreased this resistance and interacted synergistically with SHetA2 in mutant and wild type p53-expressing cell lines in association with elevated reactive oxygen species/ATP ratios. Tumor-free rates in animals were 0% (controls), 25% (PRIMA1MET ), 42% (SHetA2) and 67% (combination). SHetA2 (p = 0.004) and PRIMA1MET (p = 0.048) functioned additively in preventing tumor development with no observed toxicity. These results justify the development of SHetA2 and PRIMA-1MET alone and in combination for ovarian cancer maintenance therapy.

Studies of drug interactions with alpha1-acid glycoprotein by using on-line immunoextraction and high-performance affinity chromatography.

A method that combined on-line immunoextraction with high-performance affinity chromatography was developed to examine the binding of drugs with α1-acid glycoprotein (AGP). Affinity microcolumns containing immobilized polyclonal anti-AGP antibodies were developed that had a capture efficiency of up to 98.4% for AGP and a binding capacity of 0.72nmol AGP when using a 20mm×2.1mm i.d. microcolumn. These microcolumns were employed in various formats to examine the binding of drugs to normal AGP and AGP that had been adsorbed from serum samples for patients with systemic lupus erythematosus (SLE). Drugs that were screened in zonal elution experiments for their overall binding to these types of AGP included chlorpromazine, disopyramide, imipramine, propranolol, and warfarin. Most of these drugs showed an increase in their binding to the AGP from SLE serum when compared to normal AGP (i.e., an increase of 13-76%); however, disopyramide gave a 21-25% decrease in retention when the same AGP samples were compared. Frontal analysis was used to further evaluate the binding of disopyramide and imipramine to these forms of AGP. Both drugs gave a good fit to a model that involved a combination of saturable and non-saturable interactions with AGP. Changes in the non-saturable interactions accounted for most of variations seen in the binding of disopyramide and imipramine with the AGP samples. The methods used in this study could be adapted for use in personalized medicine and the study of other proteins or drugs using aqueous mixtures or clinical samples.

12-HETrE inhibits platelet reactivity and thrombosis in part through the prostacyclin receptor.

The dihomo-γ-linolenic acid (DGLA)-derived metabolite of 12-lipoxygenase, 12-hydroxy-eicosatrienoic acid (12-HETrE), was recently shown to potently inhibit thrombus formation without prolonging bleeding in murine models. Although 12-HETrE was found to inhibit platelet activation via the Gαs signaling pathway, the Gαs-coupled receptor by which 12-HETrE mediates its antiplatelet effects has yet to be identified. Defining the receptor by which 12-HETrE exerts its effects is key to determining its therapeutic potential as an antiplatelet drug. Therefore, the goal of this study was to determine the Gαs-coupled platelet receptor through which 12-HETrE exerts its antiplatelet effects. In this study, we showed that pharmacological inhibition of the prostacyclin (IP) receptor in human platelets or genetic ablation of IP in murine platelets prevented 12-HETrE from blocking aggregation in vitro. Furthermore, the antithrombotic effects of 12-HETrE were significantly diminished in IP knockout mice in vivo. Together these data demonstrate that the antiplatelet effects of 12-HETrE are at least partially dependent on IP signaling. Importantly, this work identified 12-HETrE as a novel regulator of IP signaling that may aid in the rationale for design of novel therapeutics to inhibit platelet function. Additionally, this study provides further insight into the mechanism by which DGLA supplementation inhibits platelets function.