2019

Podium

Chaturvedi LS and Vyas D.  Irreversible Proteasome Inhibitor Regulate CDK Inhibitor p21 & c-Jun Kinase in MDA-MB-231Cancer Cells. Presented at the Annual Academic Surgical Congress. Houston TX, February 2019.

Abstract

Introduction: It is estimated that one million cases of breast cancer are diagnosed annually worldwide. Of these, approximately 12-20% are of the triple-negative breast cancer (TNBC) that do not express receptors for estrogen (ER), progesterone (PR) or human epidermal growth factor receptor 2 (HER2). TNBC is typically treated with a combination of other therapies such as chemotherapy, radiation, and surgery. Therefore, there is urgent need of new therapy for TNBC patients. Carfilzomib (CFZ) is a selective irreversible second generation proteasome inhibitor being used for the treatment of relapsed and refractory multiple myeloma as anticancer therapy. We have previously reported antiproliferative and apoptotic effects of CFZ alone or in combination with Doxorubicin (DOX) on human TNBC-MDA-MB-231 cancer cells. Overexpression of cyclin-dependent kinase inhibitor CDKN1A/p21Waf1/Cip1 and elevated phosphorylation of stress activated c-Jun NH2-terminal kinase has been reported in TNBC with adverse pathological parameters and poor prognosis. However, the role of CFZ in the regulation of CDKN1A/p21Waf1/Cip1 protein expression and c-Jun NH2-terminal kinase activation has not been determined in human TNBC-MDA-MB-231 cancer cells. Herein, we investigated role of CFZ in the modulation of CDKN1A/p21Waf1/Cip1 and c-Jun NH2-terminal kinase activation in human TNBC-MDA-MB-231 breast cancer cells.

Methods: Human TNBC-MDA-MB-231 cell-line, a model for TNBC was treated by various concentrations of CFZ alone, in a combination with DOX or vehicle control dimethyl sulfoxide (DMSO). Cell Counting Kit-8 (CCK-8) using WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt) was used to detect cell viability. Annexin V-FITC apoptosis detection kit and flow cytometry was used to analyze the cell cycle. Western blot was used to detect expression of cyclin-dependent kinase inhibitor CDKN1A/p21Waf1/Cip1 protein and phospho specific c-Jun NH2-terminal kinase antibodies


Results: We confirmed antiproliferative and apoptotic effects of CFZ alone or in combination with DOX using CCK-8 viability and Annexin V-FITC apoptosis detection Kit respectively. The immunoblot analysis revealed that CFZ alone significantly inhibited CDKN1A/p21Waf1/Cip1 in a dose dependent manner and as well as in a combination with DOX in comparison to vehicle control DMSO. Furthermore, CFZ alone and in a combination with DOX also significantly inhibited the phosphorylation of stress activated c-Jun NH2-terminal kinase in human TNBC- MDA-MB-231 cancer cells.

Conclusion:  Our data suggests that irreversible proteasome inhibitor CFZ alone and in combination with DOX may induce apoptosis and inhibit proliferation by modulating CDKN1A/p21Waf1/Cip1 protein expression and phosphorylation of stress activated c-Jun NH2-terminal kinase in human MDA-MB-231 breast cancer cells. Further investigation will encourage potential use of CFZ alone and in combination of DOX against tumors harboring drug-resistant phenotypes.

 

Podium

Chaturvedi LS and Vyas D. Second Generation Irreversible Proteasome Inhibitor Carfilzomib (CFZ) Inhibits Proliferation and Induces Apoptosis by down-regulating cyclin-D1, CDKN1A/p21 and c-Jun NH2-terminal Kinase Activation in Human Triple-Negative Breast MDA-MB-231Cancer Cells. Presented at the Northern California Chapter of the American College of Surgeons (NCCACS) Russell Surgical Trainee Research Competition meeting. Berkeley CA, March 2019.

Abstract

Currently there is no targeted effective therapy available for triple-negative breast cancer (TNBC) patients. The overexpression of cyclin-dependent kinase inhibitor CDKN1A/p21Waf1/Cip1 and an elevated phosphorylation of stress-activated c-Jun NH2-terminal kinase have been reported in TNBC patients with poor prognosis. Carfilzomib (CFZ) is a selective second generation irreversible proteasome inhibitor being approved for treatment of relapsed and refractory multiple myeloma. However, the role of CFZ in the regulation of CDKN1A/p21Waf1/Cip1 and c-Jun NH2-terminal kinase activation has not been fully elucidated in human TNBC cells. Herein, we investigated the role of CFZ in the modulation of CDKN1A/p21Waf1/Cip1 and c-Jun NH2-terminal kinase activation in human TNBC-MDA-MB-231 cells. CFZ inhibited proliferation, induced cell-cycle alterations and increased apoptosis in a dose and time dependent manner in MDA-MB-231 cells. Its action was compared to Doxorubicin (DOX), a first line chemotherapeutic agent. Flow cytometry analysis revealed that a combination of both CFZ and DOX significantly enhanced early and late apoptosis in comparison to either drug alone. The immunoblot analysis revealed that CFZ enhanced the reduction of cyclin-D1 protein expression, p21, and phosphorylation of stress activated c-Jun NH2-terminal kinase and induction of DNA Double-strand breaks (DSBs) by increasing the histone phosphorylation gamma-H2AX (Ser139). Our data suggests that irreversible proteasome inhibitor CFZ induces antiproliferation and apoptosis by down-regulating cyclin-D1, p21 protein expression, c-Jun NH2-terminal kinase phosphorylation and enhancing the gamma-H2AX (Ser139) phosphorylation in MDA-MB-231 cells. These findings suggest a potential use of CFZ against tumors harboring drug-resistant phenotypes.

 

Poster

Asadirad M, Talbott G, Satow B, Sharma U, Woldemariam T, Malhotra A. Identifying Mitochondrial Targets for Pancreatic Cancer Employing Unconventional Combinations of Middle Eastern Phytoextracts with Raloxifene. Presented at the American Society of Pharmacology and Experimental Therapeutics (ASPET) Meeting. Orlando FL, April 2019.

Abstract

PCa has a high mortality, substantial chemoresistance, and a poor 5-year survival rate of 6 to 8%. It is the fourth leading cause of deaths due to cancer in the United States, killing over 53,000 Americans each year, and 250,000 people worldwide. Little is known about the regulation of mitophagy in PCa by drugs and dietary components. There is also a gap in knowledge concerning molecular interactions between drugs and food and the K-ras pathway in PCa. This is important because mutations in the human homologue of the Kirsten Rat Sarcoma (KRAS) viral oncogene occur in 90% of PCa patients and drive the early development of PCa. Our previous work showed that enhanced mitophagy, which prevents the accumulation of damaged mitochondria in PCa cells was alleviated by repurposing the FDA-approved selective estrogen receptor modulator raloxifene to treat PCa cells. Here, we used a combination of Middle Eastern dietary components (MEDC) and raloxifene to mitigate mitochondrial renewal and identify novel mitochondrial targets for PCa. We screened crude aqueous extracts from six spices and plants used in Middle Eastern and Indian cuisines, including artichoke (Cynara scolymus), curry leaf (Murraya koenigii), dill (Anethum graveolens), noni (Morinda citrifolia), olive (Olea europaea), rose (Rosa damascena), and sumac (Rhus coriaria) for cytotoxicity in the PCa cell lines, MIA PaCa-2 (similar KRAS gene mutation as majority of patients) and PANC-1 (less common KRAS gene mutation). Specifically, we treated PCa cells in vitro with exponentially increasing doses of aqueous extracts at 300 ng/mL, 3 µg/mL, 30 µg/mL, and 300 µg/mL for 48 hours followed by an MTT assays. Aqueous extracts of sumac and artichoke (AESA) induced maximum cytotoxicity, which was exacerbated by raloxifene (AESA-R). To address whether cytotoxicity was limited to PCa cells, we used NIH-3T3 cells as a non-cancer control, showing a sparing effect. We also tested 1:1 combinations of AESA and found evidence of activity. To identify specific compounds, using a fractional approach based on preparative HPLC, 100 mg of the residue was chromatographed onto a silica gel column and gradient eluted using dichloromethane/methanol (gradient, 0, 100%, v/v). Fractions and eluates were monitored by thin layer chromatography using precoated silica gel 60 F254, 0.25 mm aluminum backed plates. Cytotoxic fractions were reconstituted in deuterated solvents and analyzed by NMR spectroscopy to identify pentagalloyl glucose as the bioactive component in sumac. To elucidate the mechanism for the cytotoxicity, we showed that AESA and AESA-R activated caspase-3 and 7, induced mitochondrial ROS, and reduced mitochondrial membrane potential in PCa cells, starting within 20 minutes and sustained for 48 hours following treatment. Western blot showed changes in the expression of proapoptotic (bim) and the translocation of antiapoptotic proteins (Bcl2), sustained over 48 hours. Our data demonstrate for the first time that specific food-drug combinations alter mitochondrial biology and response including the regulation of mitophagy in PCa cells.

 

Poster

Malhotra A. Enhancing Team-based Learning of Pharmacology Using Technology-assisted Prerequisite Content Preparation. Presented at the American Society of Pharmacology and Experimental Therapeutics (ASPET) Meeting. Orlando FL, April 2019.

Abstract

Multiple modalities are encouraged for creating and enhancing active learning in pharmacology pedagogy in pharmacy and medical schools. Team-based learning engages students in a detailed pedagogy that begins with pre-class self-directed preparation, leading up to an in-class individual Readiness Assurance Test (iRAT), which can be set by the program to mandate a certain level of competency by individual students. Typically, the iRAT is subsequently administered to the entire team as a team Readiness Assurance Test (tRAT). Based on the iRAT performance, a short lecture is delivered, emphasizing possible areas of class deficiency identified by the iRAT. Finally, students are asked to work in teams on application exercises. Although TBL is very effective in encouraging active participation in and out of class, team dynamics and other factors may lead to grade inflation when team scores mask and enhance the performance of borderline students. The California Northstate University College of Pharmacy employs a hybrid-TBL approach to engage learners in an active learning environment, with multiple didactic courses in the first, second and third professional years (P1, P2, P3) of the Doctor of Pharmacy program. To enhance TBL delivery in our pharmacology courses, we prepared a series of video lectures using online technology to help students prepare for their iRATs and tRATs. These short “Info-Blast” video lectures were typically 30-40 minutes in length, posted to YouTube, and emphasized the salient learning objectives, goals, terminology, and concepts included in prerequisite content required for upcoming pharmacology course lectures. Lectures included Pharmacogenomics, Biotechnology and An Introduction to Viruses in the Cell and Molecular Biology course (P1), and the pathophysiology of arrhythmia, the pharmacology of antiarrhythmic drugs, dyslipidemia, and dyslipidemia drugs in the Pathophysiology and Pharmacology II, Cardiovascular Sciences course (P2). The YouTube videos were accompanied by documents detailing self-directed Learning Objectives and a Practice Question Workbook, all of which were posted at least two weeks ahead of the class. Students provided comments and suggestions verbally and via email which were strongly positive.  Student iRAT scores and exam performance scores were captured using TurningPoint and ExamSoft and showed a positive correlation with the number of hours spent watching the video lectures. Among the advantages mentioned by students was the ease of access, the ability to repeat the lecture and detailed clarity added to expectations regarding outcomes and performance. One disadvantage mentioned was the extra time needed to prepare using the enhanced content, which we hope to address by using online technology such as InsertLearning to incorporate textual sources with the posted video lectures.

California Northstate University College of Pharmacy ♦ 9700 West Taron Drive ♦ Elk Grove, CA 95757 ♦ Phone: (916) 686-7400

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