Sorafenib combined vitamin K induces apoptosis in human pancreatic cancer cell lines through RAF/MEK/ERK and c-Jun NH2-terminal kinase pathways

Abstract

Apoptosis has been shown to be induced by many agents, including the clinically useful Sorafenib and K vitamins (VKs). Since few agents have activity against pancreas cancer cell growth, we evaluated the role of naturally occurring K vitamins and Sorafenib both independently and together on the growth in culture of pancreas adenocarcinoma cell lines, including PL-5, PANC-1, and MIA PaCa-2. We found that when a K vitamin was combined with Sorafenib, the dose of Sorafenib required for growth inhibition was substantially reduced. Furthermore, growth could be inhibited at doses of each VK plus Sorafenib in combination that were ineffective when used alone. This effect was seen using vitamins K1, K2, and K5. The combination of VK1 plus Sorafenib-induced apoptosis, as determined by both FACS and TUNEL staining. Phospho-ERK and Bcl-2 levels were decreased, but not levels of other bcl-2 family members. Cleavage of caspases 3 and 8, PARP and Bid were all induced by this combination. Vitamin K1 plus Sorafenib combination also resulted in elevated levels of activated c-Jun N-terminal kinase (JNK) and its substrates c-Jun and FasL. JNK inhibition partly antagonized the induction of apoptosis. Thus, combination VK1 plus Sorafenib strongly induced growth inhibition and apoptosis in pancreas cancer cells, involving both inhibition of the RAF/MEK/ERK pathway as well as activation of the JNK, c-Jun and FasL apoptotic pathway. Since both agents are available for human use, the combination is attractive for evaluation against pancreas cancer growth in vivo.

Figure 1. Sorafenib plus vitamin K inhibits pancreas cancer cell growth

A, PL5, MIA PaCa-2 and PANC-1 cells were treated with K1 50 µM, Sorafenib 2.5 µM or K1 plus Sorafenib and the cell number was estimated by a DNA fluorometric assay. B. PL5 cells were treated separately with 50 µM K1, 25 µM K2, 10 µM K5, 2.5 µM Sorafenib or combination Sorafenib with Ks. Cell number was counted on day 3. Each experiment was run in triplet, repeated three times and expressed mean SD. P value is determined by student’s t test.

Figure 2. Combination Sorafenib and vitamin K1 induces apoptosis in pancreas cancer cell

A: TUNEL staining. PL5 cells were treated with vitamin k1 (50 µM), Sorafenib (2.5 µM), combination vitamin k1 (50 µM) and Sorafenib (2.5 µM), or pretreated with casepases inhibitor 2 hours then incubated with vitamin k1 plus Sorafenib. TUNEL staining cells were observed at 40X magnification. B. PL5 cells were treated as the same condition in Fig. A. Floating and adherent cells were harvested at 36 hours and analyzed by flow cytometry. C: Quantitative of death cells in B.

Figure 3. Inhibition of p-ERK increases caspase-8 activation

Sorafenib or vitamin k1 inhibits p-ERK in does dependent in PL5 cells (A, B). C. Lower concentration of Vitamin k1 (50 µM) plus Sorafenib (2.5 µM) inhibits RAF/MEK/ERK pathway in PL5 cells. D. PL5 cells were treated with various does of U0126 for 36 hours. Cells were lysed and 20 µg of soluble protein was separated by electrophoresis on a SDS-PAGE gel. Protein phosphorylation was detected by Western blot analysis.

Figure 3. Inhibition of p-ERK increases caspase-8 activation

Sorafenib or vitamin k1 inhibits p-ERK in does dependent in PL5 cells (A, B). C. Lower concentration of Vitamin k1 (50 µM) plus Sorafenib (2.5 µM) inhibits RAF/MEK/ERK pathway in PL5 cells. D. PL5 cells were treated with various does of U0126 for 36 hours. Cells were lysed and 20 µg of soluble protein was separated by electrophoresis on a SDS-PAGE gel. Protein phosphorylation was detected by Western blot analysis.

Figure 4. Combination vitamin k1 and Sorafenib activates the extrinsic caspase death pathway

A: Cleaved PARP and caspase-3 can be detected after treatment 36 hours with vitamin k1(50 µM) plus Sorafenib (2.5 µM) in PL5 cells; B: Combination treatment with Sorafenib and vitamin k1 does not affect the cytochrome C release from the mitochondria in PL5 cells. C: Procaspase-8 was activated and its substrate Bid was cleaved after treatment with vitamin k1 and Sorafenib in PL5 cells, but no caspase-9 activity was detected in the same lysates. Cells were lysed and 20 µg of soluble protein was separated by electrophoresis on a SDS-PAGE gel. Protein levels were detected by Western blot analysis. Actin was used as loading control.

Figure 4. Combination vitamin k1 and Sorafenib activates the extrinsic caspase death pathway

A: Cleaved PARP and caspase-3 can be detected after treatment 36 hours with vitamin k1(50 µM) plus Sorafenib (2.5 µM) in PL5 cells; B: Combination treatment with Sorafenib and vitamin k1 does not affect the cytochrome C release from the mitochondria in PL5 cells. C: Procaspase-8 was activated and its substrate Bid was cleaved after treatment with vitamin k1 and Sorafenib in PL5 cells, but no caspase-9 activity was detected in the same lysates. Cells were lysed and 20 µg of soluble protein was separated by electrophoresis on a SDS-PAGE gel. Protein levels were detected by Western blot analysis. Actin was used as loading control.

Figure 5. Sorafenib plus vitamin k1 modulates the expression of Bcl-2 family and FasL in PL5 cells

PL5 cells were plated and treated for 36 hours with vehicle, vitamin k1 (50 µM), Sorafenib (2.5 µM), and both vitamin k1 and Sorafenib. Cells were isolated and subjected to SDS-PAGE followed by immunobloting to determine the expression of Bcl-2, Mcl-1, Survivin, Bcl-xL and Bax (A); FasL, FADD (B).

Figure 6. JNK and c-Jun activation are involved in vitamin k1 plus Sorafenib induced apoptosis in PL5 cells

A. PL5 cells were treated with 50 µM vitamin k1 and 2.5 µM Sorafenib for 36 hours and levels of phospho-JNK and its key substrate phospho-c-Jun were analyzed by Western blot from whole-cell lysates. B. PL5 cells were treated with 50 µM vitamin k1 and 2.5 µM Sorafenib for 36 hours in the presence or absence of JNK inhibitor. After treatment, cells were lysed and 20 µg proteins were separated on SDS-PAGE gel or the percentage of apoptotic cells were determined by flow cytometry (C).

Figure 7

The mechanism of combination Sorafenib and vitamin k1 seems to involve inhibition of MEK/ERK activity and activation of JNK/c-Jun. Modulation of both of these pathways results in activation of caspase-8, ultimately leading to apoptotic cell death.