Pancreatic cancer is an aggressive and deadly malignancy responsible for the death of over 37,000 Americans each year. Gemcitabine-based therapy is the standard treatment for pancreatic cancer but has limited efficacy due to chemoresistance. In this study, we evaluated the in vitro and in vivo effects of gemcitabine combined with the selective nuclear export (CRM1) inhibitor KPT-330 on pancreatic cancer growth. Human pancreatic cancer MiaPaCa-2 and metastatic pancreatic cancer L3.6pl cell lines were treated with different concentrations of KPT-330 and gemcitabine alone or in combination, and anchorage–dependent/independent growth was recorded. In addition, L3.6pl cells with luciferase were injected orthotopically into pancreas of athymic nude mice, which were treated with 1) vehicle (PBS 1 mL/kg IP, 2/week and povidone/pluronic F68 1 mL/kg PO, 3/week), 2) KPT-330 (20 mg/kg PO, 3/week), 3) gemcitabine (100 mg/kg IP, 2/week), or 4) KPT-330 (10 mg/kg) + gemcitabine (50 mg/kg) for 4 weeks. KPT-330 and gemcitabine alone dose-dependently inhibited anchorage-dependent growth in vitro and tumor volume in vivo compared to vehicle treatment. However, the combination inhibited growth synergistically. In combination, KPT-330 and gemcitabine acted synergistically to enhance pancreatic cancer cell death greater than each single-agent therapy. Mechanistically, KPT-330 and gemcitabine promoted apoptosis, induced p27, depleted survivin, and inhibited accumulation of DNA repair proteins. Together, our data suggest that KPT-330 potentiates the antitumor activity of gemcitabine in human pancreatic cancer through inhibition of tumor growth, depletion of the anti-apoptotic proteins, and induction of apoptosis.
Figure 4. MK-2206 synergizes with CarboTaxol in reducing anchorage-independent growth in three gastric cancer cell lines. aGs cells treated with 100 μM MK-2206, 100 nM CarboTaxol (1000:1 molar ratio of carboplatinum and paclitaxel respectively) or combination for 2 weeks. MK-2206 was re-dosed twice weekly. *The CarboTaxol and MK-2206 combination significantly reduced colony counts relative to CarboTaxol alone (unpaired t test P values were 0.002, 0.001, and 0.03 for aGs, sNU-1, and sNU-16 cell lines, respectively).
Background: Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. Gemcitabine is currently the standard chemotherapeutic agent for advanced pancreatic cancer, but has limited efficacy due to chemoresistance and dose escalation toxicity. Therefore an effective and safer therapy is warranted against this deadly malignancy. Selective nuclear export inhibitor KPT-330 inhibits the exportin 1 (CRM1) leading to nuclear localization of tumor suppressor proteins. The aim of this study was to evaluate the combination of KPT-330 and gemcitabine on anchorage-dependent and indepencent metastatic tumor cell growth, pro and antiapoptotic protein expression in vitro and in vivo orthotopic mouse model of metastatic human pancreatic cancer. Methods: Human pancreatic cancer cell MiaPaCa-2 and human metastatic pancreatic cancer cell L3.6pl were treated with different concentrations of KPT-330 (0.1-10 μM), and gemcitabine (0.1-10 μM) alone and in combination and anchorage-dependent growth recorded using MTT assay for 72 h. The effect of KPT330 and gemcitabine alone and in combination on anchorage independent growth were performed using soft agar colony formation assay. Human metastatic pancreatic cancer cells L3.6pl with luciferase were injected orthotopically into pancreas of female athymic nude mice and were treated with 1) Vehicle (PBS 1 ml/kg, ip, 2/week and PVP/PF68 1ml/kg, po, 3/week), 2) KPT330 (20 mg/kg, po, 3/week), 3) gemcitabine (100 mg/kg, ip, 2/week) and 4) KPT330 (10 mg/kg, po, 3/week + gemcitabine (50 mg/kg, ip, 2/week) for 4 weeks. The tumor volume and tumor weights were recorded after 4 weeks of treatment. The CRM1, p53, proapoptotic Bax and antiapoptotic survivin protein expressions in cells and tumor tissues were determined by Western blotting. Results: KPT-330 and gemcitabine alone significantly inhibited the anchorage-dependent growth in a concentration dependent manner. The combination of the two drugs inhibited the growth synergistically and almost completely inhibited the malignant transformation of anchorage independent cell growth. KPT-330 and gemcitabine alone decreased tumor volume and tumor weight compared to vehicle (p<0.05) but when combined together significantly decreased tumor size and weight (P<0.001). KPT-330 and gemcitabine alone and in combination induced apoptosis (Bax expression). KPT-330 but not gemcitabine depleted CRM1 expression whereas no significant change was observed in mutant p53 expression with any treatment. KPT330 and gemcitabine alone moderately depleted survivin expression compared to vehicle but when combined together, they markedly depleted survivin expression. Conclusion: KPT-330 potentiates antitumor activity of gemcitabine through inhibition of metastatic pancreatic tumor growth, depletion of the antiapoptotic protein and induction of apoptosis in human metastatic pancreatic cancer. Citation Format: Sabiha Kazim, Mokenge P. Malafa, Kazim Husain, Michael Kauffman, Shacham Sharon, Amit Mahipal. Selective nuclear export inhibitor kpt330 enhances the antitumor activity of gemcitabine in human pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1696. doi:10.1158/1538-7445.AM2014-1696
Background: We have shown that natural vitamin E γ-tocotrienol is the most bioactive tocotrienols in inhibiting the cell proliferation and induction of apoptosis in pancreatic cancer cells as well as in xenograft models. Therefore in this study we evaluated the chronic 12-month feeding of γ-tocotrienol on the prevention of the progression of PanIN lesions in a conditional KrasG12D mouse model. Methods: Offspring of LSLKRASG12D x PDX-1-Cre intercrosses (double positive by genotyping) were randomized to three groups: 1) No treatment (NT), 2) Vehicle (ethanol extracted olive oil, 1.0 ml/kg x 2/ day, PO) and 3) γ-tocotrienol (200 mg/kg x 2/day, PO). The treatment was started at the age of 10 weeks and continued for 12 months. The survival of the mice in three groups was plotted by Kaplan-Meir graph. The pancreas of KRASG12D; PDX-1-Cre mice was analyzed for the presence of murine PanINs. The apoptosis, cell cycle and signaling markers were determined by Western blotting and immunostaining. Results: No significant difference in food intake and body weight gain between γ-tocotrienol and control groups was observed during 12 months feeding. γ-tocotrienol treatment significantly increased survival of mice compared to control groups (p<0.025). Mice fed γ-tocotrienol had a significant delay in the progression of mPanIN lesions. The proportion of mPanIN-3 lesion was significantly decreased (P<0.001), whereas the relative numbers of all other lesions combined were slightly increased in the γ-tocotrienol-fed animals. γ-tocotrienol feeding also inhibited the pMEK and pERK signaling and increased p27Kip1 expression in the pancreatic tumor tissues. The apoptosis markers such as caspase-3, PARP1 cleavage, and Bax expression were increased in tumor tissues of mice treated with γ-tocotrienol compared to controls. NF-κB/p65 and the NF-κB transcriptional targets Bcl-XL was down-regulated by γ-tocotrienol treatment. The ratio of the proapoptotic (Bax) and antiapoptotic (Bcl-XL) proteins was increased almost 4-folds in the pancreatic tumor tissues compared to controls. Conclusion: γ-tocotrienol delays the progression of pancreatic cancer precursor lesions in a preclinical animal model hence warrant it's clinical use for the prevention and treatment of pancreatic cancer in high-risk patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1613. doi:1538-7445.AM2012-1613
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