Selective Nuclear Export Inhibitor KPT-330 Enhances the Antitumor Activity of Gemcitabine in Human Pancreatic Cancer

Sabiha Kazim, Mokenge P Malafa, Domenico Coppola, Kazim Husain, Sherma Zibadi, Trinayan Kashyap, Marsha Crochiere, Yosef Landesman, Tami Rashal, Daniel M Sullivan, Amit Mahipal, Sabiha Kazim, Mokenge P Malafa, Domenico Coppola, Kazim Husain, Sherma Zibadi, Trinayan Kashyap, Marsha Crochiere, Yosef Landesman, Tami Rashal, Daniel M Sullivan, Amit Mahipal

Abstract

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 the pancreas of athymic nude mice, which were treated with (i) vehicle (PBS 1 mL/kg i.p., 2/week and povidone/pluronic F68 1 mL/kg p.o., 3/week), (ii) KPT-330 (20 mg/kg p.o., 3/week), (iii) gemcitabine (100 mg/kg i.p., 2/week), or (iv) 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 with 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 antiapoptotic proteins, and induction of apoptosis.

Conflict of interest statement

Conflict of Interests: Trinayan Kashyap, Marsha Crochiere, Yosef Landesman, and Tami Rashal are employees of Karyopharm Therapeutics Inc.

©2015 American Association for Cancer Research.

Figures

Fig. 1
Fig. 1
A, Effect of gemcitabine (Gem) and KPT-330 alone and in combination on pancreatic cancer cell proliferation (MTT assay). Gemcitabine and KPT-330 significantly (aP < 0.001,aP<0.01) decreased proliferation of MiaPaCa-2 and L3.6pl cells versus vehicle, with the combination being more effective in inhibiting cell proliferation than either drug alone (bP < 0.05). Points, means; bars, SE (n = 3–5). All statistical analyses were performed using ANOVA with Duncan test. B, Isobologram with combination index (CI) showing synergistic effect of gemcitabine plus KPT-330 (1:1; G/K) in MiaPaCa-2 and L3.6pl cells.
Fig. 2
Fig. 2
A, Effect of gemcitabine and KPT-330 alone and in combination on pancreatic cancer malignant transformation in MiaPaCa-2 and L3.6pl cells. V, vehicle. B, Gemcitabine and KPT-330 alone significantly inhibited colony formation (*P < 0.01, aP<0.02), with combination resulting in almost complete inhibition of the malignant transformation versus vehicle (**P<0.001, bP<0.001) or versus gemcitabine or KPT-330 alone (#P < 0.001, cP<0.01). Bars (means) ± SE (n = 3). All statistical analyses were performed using ANOVA with Duncan test.
Fig. 3
Fig. 3
A, Effect of gemcitabine and KPT-330 alone and in combination on pancreatic cancer survival (Trypan blue) in MiaPaCa-2 and L3.6pl cells. Both gemcitabine and KPT-330 significantly induced cell death compared to vehicle (aP < 0.001). However, the combination resulted in greater effect on cell death than vehicle alone (aP < 0.001) or gemcitabine or KPT-330 alone (bP < 0.05). Bars (means) ± SE (n = 3–5). All statistical analyses were performed using ANOVA with Duncan test. B, Western blot of CRM1, cleaved PARP, p27, Bax, and survivin and densitometry of the protein bands in MiaPaCa-2 cells. KPT-330 but not gemcitabine almost completely and significantly (aP<0.001) depleted CRM1 protein expression in MiaPaCa-2 cells compared to vehicle, with the combination depleting CRM1 expression significantly (bP<0.05). Gemcitabine and KPT-330 alone significantly (bP<0.05 and aP<0.001) induced p27 and Bax expression, whereas the combination eliciting greater induction than either drug alone. Gemcitabine and KPT-330 alone significantly (aP<0.001 and bP<0.05) depleted survivin expression, whereas the combination had greater depletion than either drug alone. Gemcitabine and KPT-330 alone and in combination induced apoptosis (cleaved PARP) in these cells. Points, means; bars, SE (n = 3). C, Western blot of CRM1, cleaved PARP, p27, Bax, and survivin and densitometry of the protein bands in L3.6pl cells. KPT-330 but not gemcitabine significantly (aP<0.001) depleted CRM1 protein expression in L3.6pl cells versus vehicle, with combination also significantly depleting CRM1 expression (aP<0.001). Gemcitabine and KPT-330 alone and in combination significantly (bP<0.05 and aP<0.001) induced p27 and Bax expression in these cells. Gemcitabine and KPT-330 alone significantly (aP<0.001) depleted survivin expression, with the combination having greater depletion than either drug alone. Gemcitabine and KPT-330 alone and in combination induced apoptosis (cleaved PARP) in these cells. Points, means; bars, SE (n = 3). All statistical analyses were performed using ANOVA with Duncan test.
Fig. 4
Fig. 4
A, Western blot showing effect of KPT-330 on DNA damage repair proteins RAD51, Chk1, PMS2, MLH1, and cleaved caspase-3 after 24- and 48-hour incubation in MiaPaCa-2 cells. KPT-330 reduced steady-state levels of DNA damage repair proteins in a dose- and time-dependent manner. B, Western blot showing effect of gemcitabine and KPT-330 alone and in combination on DNA damage repair proteins in MiaPaCa-2 cells after 24 hours. KPT-330 abrogated activation through phosphorylation of Chk1 protein after gemcitabine treatment. In addition, the combination enhanced apoptosis, marked by increased caspase-3 cleavage. C, Immunofluorescence of gamma-H2AX phosphorylation in MiaPaCa-2 cells by confocal microscopy (green fluorescence) was higher with KPT-330 + gemcitabine.
Fig. 5
Fig. 5
A, Effect of gemcitabine and KPT-330 alone and in combination on orthotopic pancreatic L3.6pl tumor growth in mice. Gemcitabine or KPT-330 significantly reduced tumor volume (aP<0.05) compared to vehicle, with the combination resulting in more significantly reduced tumor volume compared to vehicle (aP<0.001) or either drug alone (P<0.01 and P<0.05). Results are means and SE (n=5). B, Mean pancreatic tumor weight changes in mice after drug treatment. Gemcitabine and KPT-330 significantly reduced tumor weight (bP<0.05) compared to vehicle, with their combination eliciting a more significant reduction versus vehicle (aP<0.001) or either drug alone (cP<0.05). Bars indicate SE (n=5). All statistical analyses were performed using ANOVA with Duncan test. C, Liver metastasis changes in mice after drug treatment. Gemcitabine and KPT-330 significantly reduced liver metastasis (aP<0.05 and bP<0.01, respectively) compared to vehicle. However, the combination of the 2 drugs more significantly reduced liver metastasis compared to vehicle (aP<0.01) or compared to gemcitabine or KPT-330 alone (cP<0.02). Bars indicate SE (n=5). All statistical analyses were performed using ANOVA with Duncan test. D, Tumor apoptosis (TUNEL) immunofluorescence staining changes in mice after drug treatment. Gemcitabine and KPT-330 induced apoptosis in tumor compared to vehicle. However the combination of the 2 drugs induced more apoptosis than either drug alone in tumor tissues of mice.
Fig. 6
Fig. 6
A, Effect of gemcitabine and KPT-330 alone and in combination on pancreatic tumor cell proliferation index (Ki-67 immunostaining) in mice. Both gemcitabine and KPT-330 significantly inhibited tumor cell growth compared to vehicle (bP<0.05 and aP<0.001). However, the combination resulted in greater inhibition of tumor cell growth versus vehicle (aP < 0.001) or either drug alone (cP < 0.02). Bars (means) ± SE (n=5). B, KPT-330 more significantly increased p27 nuclear immunostaining (bP<0.001) than gemcitabine (aP<0.01) compared to vehicle, with the combination having a greater significant effect versus vehicle (bP<0.001) or either drug alone (cP<0.02). Bars (means) ± SE (n=5). C, KPT-330 more significantly decreased survivin immunostaining (aP<0.001) than gemcitabine (bP<0.05) compared to vehicle, with the combination having a greater significant effect versus vehicle (aP<0.001) or either drug alone (cP < 0.02). Bars (means) ± SE (n=5). D, Gemcitabine more significantly increased cleaved caspase-3 immunostaining (aP<0.001) than KPT-330 (bP<0.01) compared to vehicle, with the combination having a greater significant effect versus vehicle (aP<0.001) or either drug alone (cP < 0.01). Bars (means) ± SE (n=5). All statistical analyses were performed using ANOVA with Duncan test. E, Western blot of CRM1, p27, Bax, and survivin expression in orthotopic pancreatic L3.6pl tumor tissues of mice and densitometry of protein bands. KPT-330 but not gemcitabine significantly (aP<0.001) depleted CRM1 protein expression compared to vehicle. However, the combination resulted in a more depletion of CRM1 in tumor tissues than either drug alone. Gemcitabine and KPT-330 alone significantly (bP<0.05 and aP<0.001) induced p27 and Bax expression, whereas the combination elicited greater induction than either drug alone. Gemcitabine and KPT-330 alone significantly (bP<0.05) depleted survivin expression, whereas the combination had greater depletion than either drug alone. Bars (means) ± SE (n=3). All statistical analyses were performed using ANOVA with Duncan test.

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