Sorafenib enhances pemetrexed cytotoxicity through an autophagy-dependent mechanism in cancer cells

Abstract

Pemetrexed (ALIMTA, Lilly) is a folate antimetabolite that has been approved by the U.S. Food and Drug Administration for the treatment of non-small cell lung cancer and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multikinase inhibitor sorafenib (Nexavar, Bayer), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K, and/or phosphorylated mTOR, in addition to class III receptor tyrosine kinases such as platelet-derived growth factor receptor beta and VEGF receptors, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.

Figure 1. Pemetrexed induces autophagy and tumor cell killing that is suppressed by knock down of Beclin1 or treatment with 3-methyl adenine

Panel A. H460 and 4T1 cells were transfected with a plasmid to express LC3-GFP. Twenty four h after transfection cells were treated with vehicle (PBS) or pemetrexed (0.03–3.0 µM). Cells were examined under a fluorescent microscope (X40) at the indicated times after drug exposure and the mean number of vesicles in 40 selected random cells in triplicate wells calculated (n = 2 studies, +/− SEM; * p < 0.05 greater than vehicle control value). Upper inset: H460 cells, 12h after pemetrexed treatment (0.3 µM) treatment were isolated and immunoblotting performed to determine the expression and lipidation / conversion of LC3 to LC3II. Panels B and C. H460, 4T1, BT474 and HuH7 cells as indicated are transfected with siRNAs (si-scramble, siSCR; siBeclin1; 20 nM). Twenty four h after transfection cells were treated with vehicle (PBS) or pemetrexed (0.03–3.0 µM). As indicated, just prior to pemetrexed treatment, cells were also treated with vehicle (PBS) or with 3-methyl adenine (1 mM). Cells were isolated 24h after treatment and viability determined by annexin-PI staining / flow cytometry (n = 3, +/− SEM; # p < 0.05 greater than corresponding vehicle treated or siSCR transfected cell value). Data in Panel C is presented without the control “1.00” survival bars for clarity.

Figure 1. Pemetrexed induces autophagy and tumor cell killing that is suppressed by knock down of Beclin1 or treatment with 3-methyl adenine

Panel A. H460 and 4T1 cells were transfected with a plasmid to express LC3-GFP. Twenty four h after transfection cells were treated with vehicle (PBS) or pemetrexed (0.03–3.0 µM). Cells were examined under a fluorescent microscope (X40) at the indicated times after drug exposure and the mean number of vesicles in 40 selected random cells in triplicate wells calculated (n = 2 studies, +/− SEM; * p < 0.05 greater than vehicle control value). Upper inset: H460 cells, 12h after pemetrexed treatment (0.3 µM) treatment were isolated and immunoblotting performed to determine the expression and lipidation / conversion of LC3 to LC3II. Panels B and C. H460, 4T1, BT474 and HuH7 cells as indicated are transfected with siRNAs (si-scramble, siSCR; siBeclin1; 20 nM). Twenty four h after transfection cells were treated with vehicle (PBS) or pemetrexed (0.03–3.0 µM). As indicated, just prior to pemetrexed treatment, cells were also treated with vehicle (PBS) or with 3-methyl adenine (1 mM). Cells were isolated 24h after treatment and viability determined by annexin-PI staining / flow cytometry (n = 3, +/− SEM; # p < 0.05 greater than corresponding vehicle treated or siSCR transfected cell value). Data in Panel C is presented without the control “1.00” survival bars for clarity.

Figure 2. Pemetrexed interacts with sorafenib in a dose-dependent fashion to increase autophagy and tumor cell killing that is suppressed by knock down of Beclin1

Panel A. BT474, 4T1 and HuH7 cells as indicated are transfected with siRNAs (si-scramble, siSCR; siBeclin1; 20 nM) and with a plasmid to express LC3-GFP. Twenty four h after transfection cells were treated with vehicle (PBS) or pemetrexed (0.03–3.0 µM) and/or vehicle (DMSO) or sorafenib. Twelve h after drug exposure cells were examined under a fluorescent microscope (X40) at the indicated times after drug exposure and the mean number of vesicles in 40 random cells in triplicate calculated per experiment (n = 2 studies, +/− SEM; * p < 0.05 less than corresponding siSCR value). Panel B. HuH7 and H460 cells were treated as indicated with vehicle (PBS) or pemetrexed and/or vehicle (DMSO) or sorafenib. Viability was determined in triplicate 24h later by trypan blue exclusion (HuH7, H460) or annexin-PI staining, as noted in the panel (n = 2, +/− SEM; ¶ p < 0.05 greater than vehicle control value). Panel C. Parental MCF7 and fluvestrant resistant MCF7 cells (MCF7F) were treated as indicated with vehicle (PBS) or pemetrexed and/or vehicle (DMSO) or sorafenib. Viability was determined in triplicate 24h later by trypan blue exclusion (n = 2, +/− SEM; ¶ p < 0.05 greater than vehicle control value; ¶¶ p < 0.05 greater than corresponding value in parental MCF7 cells). Panel D. BT474 and HuH7 cells as indicated are transfected with siRNAs (si-scramble, siSCR; siBeclin1; 20 nM). Twenty four h after transfection in triplicate cells were treated with vehicle (PBS) or pemetrexed (0.03–3.0 µM) and/or vehicle (DMSO) or sorafenib. Viability was determined in triplicate 24h later by trypan blue exclusion (n = 2, +/− SEM; * p < 0.05 less than corresponding siSCR value).

Figure 2. Pemetrexed interacts with sorafenib in a dose-dependent fashion to increase autophagy and tumor cell killing that is suppressed by knock down of Beclin1

Panel A. BT474, 4T1 and HuH7 cells as indicated are transfected with siRNAs (si-scramble, siSCR; siBeclin1; 20 nM) and with a plasmid to express LC3-GFP. Twenty four h after transfection cells were treated with vehicle (PBS) or pemetrexed (0.03–3.0 µM) and/or vehicle (DMSO) or sorafenib. Twelve h after drug exposure cells were examined under a fluorescent microscope (X40) at the indicated times after drug exposure and the mean number of vesicles in 40 random cells in triplicate calculated per experiment (n = 2 studies, +/− SEM; * p < 0.05 less than corresponding siSCR value). Panel B. HuH7 and H460 cells were treated as indicated with vehicle (PBS) or pemetrexed and/or vehicle (DMSO) or sorafenib. Viability was determined in triplicate 24h later by trypan blue exclusion (HuH7, H460) or annexin-PI staining, as noted in the panel (n = 2, +/− SEM; ¶ p < 0.05 greater than vehicle control value). Panel C. Parental MCF7 and fluvestrant resistant MCF7 cells (MCF7F) were treated as indicated with vehicle (PBS) or pemetrexed and/or vehicle (DMSO) or sorafenib. Viability was determined in triplicate 24h later by trypan blue exclusion (n = 2, +/− SEM; ¶ p < 0.05 greater than vehicle control value; ¶¶ p < 0.05 greater than corresponding value in parental MCF7 cells). Panel D. BT474 and HuH7 cells as indicated are transfected with siRNAs (si-scramble, siSCR; siBeclin1; 20 nM). Twenty four h after transfection in triplicate cells were treated with vehicle (PBS) or pemetrexed (0.03–3.0 µM) and/or vehicle (DMSO) or sorafenib. Viability was determined in triplicate 24h later by trypan blue exclusion (n = 2, +/− SEM; * p < 0.05 less than corresponding siSCR value).

Figure 3. Fulvestrant resistant MCF7 cells express higher levels of autophagy markers and mitochondrial protective proteins; protection of the mitochondria blocks pemetrexed + sorafenib toxicity

Panel A. MCF7 and MCF7F cells, 24h after plating, were treated with vehicle (PBS) or pemetrexed (1.0 µM) and/or vehicle (DMSO) or sorafenib (3.0 µM). Cells were isolated 24h after drug exposure and subjected to SDS PAGE and immunoblotting against the indicated proteins as described in the Methods section. The intensity of immunostaining was normalized to either GAPDH for proteins or for phospho-proteins to the dephosphorylated protein; these values were then arbitrarily normalized with the intensity value for each protein or phosphoprotein equivalent in vehicle treated MCF7 cells to 1.00 (n = 3 +/− SEM; * p < 0.05 less than corresponding vehicle control value; @ p < 0.05 greater than corresponding vehicle control value in parental MCF7; # p < 0.05 greater than corresponding vehicle control value). Panel B. MCF7 and MCF7F cells were infected with either empty vector virus (Ad.cmv); with a virus to express dominant negative caspase 9 (Ad.dnCasp9); or with viruses to express a BCL-XL or c-FLIP-s (Ad.BCL-XL; Ad.c-FLIP). Twenty four h after infection cells were treated with vehicle (PBS) or pemetrexed (0.03–3.0 µM) and/or vehicle (DMSO) or sorafenib. Viability was determined in triplicate 48h later by trypan blue exclusion (n = 2, +/− SEM; * p < 0.05 less than corresponding vehicle control value).

Figure 4. Knock down of PDGFRβ, mTOR or p70 S6K enhances pemetrexed toxicity and inhibition of ERK1/2 suppresses drug combination toxicity

Panel A. Tumor cells, as indicated, growing in log phase were isolated 24h after plating and subjected to SDS PAGE and immunoblotting against the indicated proteins as described in the Methods section. The intensity of immunostaining was normalized to either GAPDH for proteins or for phospho-proteins to the equivalent dephosphorylated protein; these values were then normalized with the intensity value of each protein / phosphor-protein in BT474 cells defined as 1.00 (+/− SEM, n = 3) (# p < 0.05 greater than value in BT474; @ p < 0.05 greater than value in parental MCF7 and BT474; + p < 0.05 greater than value in parental MCF7. Panel B. MCF7 and MCF7F cells, 24h after plating, were treated with vehicle (PBS) or pemetrexed (1.0 µM) and/or vehicle (DMSO) or sorafenib (3.0 µM). Cells were isolated 24h after drug exposure and subjected to SDS PAGE and immunoblotting against the indicated proteins as described in the Methods section. The intensity of immunostaining was normalized to either GAPDH for proteins or for phospho-proteins to the dephosphorylated protein; these values were then arbitrarily normalized with the intensity value for each protein or phospho-protein equivalent in vehicle treated MCF7 cells to 1.00 (n = 3 +/− SEM; * p < 0.05 less than corresponding vehicle control value; @ p < 0.05 greater than corresponding vehicle control value in parental MCF7; # p < 0.05 greater than corresponding vehicle control value). Panel C. MCF7 and MCF7F cells are transfected in triplicate with siRNAs (si-scramble, siSCR; PDGFRβ; 20 nM). Twenty four h after transfection cells were treated with vehicle (PBS) or pemetrexed (0.1–1.0 µM). Viability was determined in triplicate 48h later by trypan blue exclusion (n = 2, +/− SEM; ¶ p < 0.05 greater than corresponding siSCR value). Upper immunoblots: siRNA transfected cells are treated with drugs and isolated 12h after drug treatment and immunoblotting performed for levels of SRC Y416 and PDGFRβ. Panel D. MCF7 and MCF7F cells are transfected with siRNAs (si-scramble, siSCR; si-mTOR; si-p70 S6K; 20 nM). Twenty four h after transfection cells were treated with vehicle (PBS) or pemetrexed (0.1–1.0 µM). Viability was determined in triplicate 48h later by trypan blue exclusion (n = 2, +/− SEM; ¶ p < 0.05 greater than corresponding siSCR value). Panel E. MCF7 and MCF7F cells were transfected with either empty vector plasmid (CMV) or with plasmids to express a constitutively activated form of p70 S6K and/or a constitutively activated form of mTOR. Twenty four h after transfection cells were treated with vehicle (PBS) or pemetrexed (0.03–3.0 µM) and/or vehicle (DMSO) or sorafenib. Viability was determined in triplicate 48h later by trypan blue exclusion (n = 3, +/− SEM; * p < 0.05 less than corresponding CMV value; # p < 0.05 less than difference in corresponding ca-mTOR or ca-p70 S6K).

Figure 4. Knock down of PDGFRβ, mTOR or p70 S6K enhances pemetrexed toxicity and inhibition of ERK1/2 suppresses drug combination toxicity

Panel A. Tumor cells, as indicated, growing in log phase were isolated 24h after plating and subjected to SDS PAGE and immunoblotting against the indicated proteins as described in the Methods section. The intensity of immunostaining was normalized to either GAPDH for proteins or for phospho-proteins to the equivalent dephosphorylated protein; these values were then normalized with the intensity value of each protein / phosphor-protein in BT474 cells defined as 1.00 (+/− SEM, n = 3) (# p < 0.05 greater than value in BT474; @ p < 0.05 greater than value in parental MCF7 and BT474; + p < 0.05 greater than value in parental MCF7. Panel B. MCF7 and MCF7F cells, 24h after plating, were treated with vehicle (PBS) or pemetrexed (1.0 µM) and/or vehicle (DMSO) or sorafenib (3.0 µM). Cells were isolated 24h after drug exposure and subjected to SDS PAGE and immunoblotting against the indicated proteins as described in the Methods section. The intensity of immunostaining was normalized to either GAPDH for proteins or for phospho-proteins to the dephosphorylated protein; these values were then arbitrarily normalized with the intensity value for each protein or phospho-protein equivalent in vehicle treated MCF7 cells to 1.00 (n = 3 +/− SEM; * p < 0.05 less than corresponding vehicle control value; @ p < 0.05 greater than corresponding vehicle control value in parental MCF7; # p < 0.05 greater than corresponding vehicle control value). Panel C. MCF7 and MCF7F cells are transfected in triplicate with siRNAs (si-scramble, siSCR; PDGFRβ; 20 nM). Twenty four h after transfection cells were treated with vehicle (PBS) or pemetrexed (0.1–1.0 µM). Viability was determined in triplicate 48h later by trypan blue exclusion (n = 2, +/− SEM; ¶ p < 0.05 greater than corresponding siSCR value). Upper immunoblots: siRNA transfected cells are treated with drugs and isolated 12h after drug treatment and immunoblotting performed for levels of SRC Y416 and PDGFRβ. Panel D. MCF7 and MCF7F cells are transfected with siRNAs (si-scramble, siSCR; si-mTOR; si-p70 S6K; 20 nM). Twenty four h after transfection cells were treated with vehicle (PBS) or pemetrexed (0.1–1.0 µM). Viability was determined in triplicate 48h later by trypan blue exclusion (n = 2, +/− SEM; ¶ p < 0.05 greater than corresponding siSCR value). Panel E. MCF7 and MCF7F cells were transfected with either empty vector plasmid (CMV) or with plasmids to express a constitutively activated form of p70 S6K and/or a constitutively activated form of mTOR. Twenty four h after transfection cells were treated with vehicle (PBS) or pemetrexed (0.03–3.0 µM) and/or vehicle (DMSO) or sorafenib. Viability was determined in triplicate 48h later by trypan blue exclusion (n = 3, +/− SEM; * p < 0.05 less than corresponding CMV value; # p < 0.05 less than difference in corresponding ca-mTOR or ca-p70 S6K).

Figure 4. Knock down of PDGFRβ, mTOR or p70 S6K enhances pemetrexed toxicity and inhibition of ERK1/2 suppresses drug combination toxicity

Panel A. Tumor cells, as indicated, growing in log phase were isolated 24h after plating and subjected to SDS PAGE and immunoblotting against the indicated proteins as described in the Methods section. The intensity of immunostaining was normalized to either GAPDH for proteins or for phospho-proteins to the equivalent dephosphorylated protein; these values were then normalized with the intensity value of each protein / phosphor-protein in BT474 cells defined as 1.00 (+/− SEM, n = 3) (# p < 0.05 greater than value in BT474; @ p < 0.05 greater than value in parental MCF7 and BT474; + p < 0.05 greater than value in parental MCF7. Panel B. MCF7 and MCF7F cells, 24h after plating, were treated with vehicle (PBS) or pemetrexed (1.0 µM) and/or vehicle (DMSO) or sorafenib (3.0 µM). Cells were isolated 24h after drug exposure and subjected to SDS PAGE and immunoblotting against the indicated proteins as described in the Methods section. The intensity of immunostaining was normalized to either GAPDH for proteins or for phospho-proteins to the dephosphorylated protein; these values were then arbitrarily normalized with the intensity value for each protein or phospho-protein equivalent in vehicle treated MCF7 cells to 1.00 (n = 3 +/− SEM; * p < 0.05 less than corresponding vehicle control value; @ p < 0.05 greater than corresponding vehicle control value in parental MCF7; # p < 0.05 greater than corresponding vehicle control value). Panel C. MCF7 and MCF7F cells are transfected in triplicate with siRNAs (si-scramble, siSCR; PDGFRβ; 20 nM). Twenty four h after transfection cells were treated with vehicle (PBS) or pemetrexed (0.1–1.0 µM). Viability was determined in triplicate 48h later by trypan blue exclusion (n = 2, +/− SEM; ¶ p < 0.05 greater than corresponding siSCR value). Upper immunoblots: siRNA transfected cells are treated with drugs and isolated 12h after drug treatment and immunoblotting performed for levels of SRC Y416 and PDGFRβ. Panel D. MCF7 and MCF7F cells are transfected with siRNAs (si-scramble, siSCR; si-mTOR; si-p70 S6K; 20 nM). Twenty four h after transfection cells were treated with vehicle (PBS) or pemetrexed (0.1–1.0 µM). Viability was determined in triplicate 48h later by trypan blue exclusion (n = 2, +/− SEM; ¶ p < 0.05 greater than corresponding siSCR value). Panel E. MCF7 and MCF7F cells were transfected with either empty vector plasmid (CMV) or with plasmids to express a constitutively activated form of p70 S6K and/or a constitutively activated form of mTOR. Twenty four h after transfection cells were treated with vehicle (PBS) or pemetrexed (0.03–3.0 µM) and/or vehicle (DMSO) or sorafenib. Viability was determined in triplicate 48h later by trypan blue exclusion (n = 3, +/− SEM; * p < 0.05 less than corresponding CMV value; # p < 0.05 less than difference in corresponding ca-mTOR or ca-p70 S6K).

Figure 5. Pemetrexed and sorafenib interact to suppress breast cancer tumor growth in orthotopic human and rodent syngeneic model systems

Panel A. BT474 tumors were established in the 4^th mammary fat pad of athymic mice (~75 mm³). Animals were treated with vehicle, sorafenib (sor), pemetrexed (ptx) or both drugs simultaneously as described in Methods. Animals were treated for 5 days with drugs and tumor volumes measured every two-three days as indicated, and mean tumor volumes plotted (n = 2 studies; 8 animals per group total +/− SEM; * p < 0.05 less than corresponding vehicle control value; % p < 0.05 less than corresponding sorafenib value). Panel B. BT474 tumors fourteen days after drug exposure were fixed, sectioned and stained as described in Methods. Ki67 measures tumor cell proliferative rate; TUNEL and cleaved caspase 3 the levels of tumor cell apoptosis within the tumors. Panel C. 4T1 tumors were injected into the 4^th mammary fat pad of BALB/c mice. Five days after implantation the animals were administered vehicle, sorafenib (sor), pemetrexed (ptx) or both drugs simultaneously as described in Methods for 5 days. The volumes of the tumors in each group were calculated 14 days after the final drug treatment (n = 2 studies; 8 animals per group total +/− SEM; * p < 0.05 less than ptx or sor treated tumor values).

Figure 5. Pemetrexed and sorafenib interact to suppress breast cancer tumor growth in orthotopic human and rodent syngeneic model systems

Panel A. BT474 tumors were established in the 4^th mammary fat pad of athymic mice (~75 mm³). Animals were treated with vehicle, sorafenib (sor), pemetrexed (ptx) or both drugs simultaneously as described in Methods. Animals were treated for 5 days with drugs and tumor volumes measured every two-three days as indicated, and mean tumor volumes plotted (n = 2 studies; 8 animals per group total +/− SEM; * p < 0.05 less than corresponding vehicle control value; % p < 0.05 less than corresponding sorafenib value). Panel B. BT474 tumors fourteen days after drug exposure were fixed, sectioned and stained as described in Methods. Ki67 measures tumor cell proliferative rate; TUNEL and cleaved caspase 3 the levels of tumor cell apoptosis within the tumors. Panel C. 4T1 tumors were injected into the 4^th mammary fat pad of BALB/c mice. Five days after implantation the animals were administered vehicle, sorafenib (sor), pemetrexed (ptx) or both drugs simultaneously as described in Methods for 5 days. The volumes of the tumors in each group were calculated 14 days after the final drug treatment (n = 2 studies; 8 animals per group total +/− SEM; * p < 0.05 less than ptx or sor treated tumor values).