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Frontiers in Biology

ISSN 1674-7984

ISSN 1674-7992(Online)

CN 11-5892/Q

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Front. Biol.    2017, Vol. 12 Issue (6) : 421-429    https://doi.org/10.1007/s11515-017-1475-x
RESEARCH ARTICLE
Midostaurin inhibits hormone-refractory prostate cancer PC-3 cells by modulating nPKCs and AP-1 transcription factors and their target genes involved in cell cycle
Kavya Krishnappa(), Naveen Kumar Mallesh, Srikantaradhya Chidananda Sharma, Doddamane Manjulakumari()
Department of Microbiology and Biotechnology, Bangalore University, Jnana Bharathi, Bengaluru-560 056, Karnataka, India
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Abstract

BACKGROUND: The development of prostate cancer from a clinically localized, hormone-naive state to a hormone-refractory phenotype involves a complex interplay of protein kinase C (PKC) and activator protein-1 (AP-1). Therefore, the present study aimed to uncover the roles of PKC and AP-1 through midostaurin-mediated regulation—a multi-target protein kinase inhibitor.

METHODS: Androgen Receptor-negative, hormone-refractory prostate cancer cells (PC-3) were used as an in-vitro model system. The effect of midostaurin on cell viability was assessed by an MTT assay. Expression studies on PKC-α, PKC- d, different AP-1 transcription factors, and AP-1 regulating genes were analyzed by semiquantitative RT-PCR, and protein levels of Bcl-2 were evaluated by western blotting.

RESULTS: Midostaurin decreased the viability of hormone-refractory PC-3 cells. Furthermore, midostaurin significantly induced the transcripts of apoptotic-mediated PKC- d, tumor suppressor p53, cell cycle inhibitor p21cip1/waf1, death receptor TNF-α, pro-apoptotic Bax, and Caspase-8, and eventually inhibited the expression of pro-survival PKC-ε, pro-oncogene c-Jun, c-Fos, Fra-1, positive growth regulator cyclin D1, and anti-apoptotic Bcl-2. In addition, midostaurin also decreased the protein expression of anti-apoptotic Bcl-2.

CONCLUSION: The present study provided evidence that midostaurin suppresses tumor growth and induces apoptosis in hormone-refractory PC-3 cells via modulation of PKC- d and PKC-ε expression, and regulation of PMA-altered c-Jun, c-Fos, and Fra-1 AP-1 transcription factors and their target genes involved in cell cycle regulation (cyclin D1, p53, p21, Bcl-2, and TNF-α). Thus, pharmacological targeting of PKC and AP-1 factors may have therapeutic potential against hormone-refractory prostate cancer.
Keywords protein kinase C      AP-1 factors      midostaurin      semi-qRT-PCR      western blotting     
Corresponding Author(s): Kavya Krishnappa,Doddamane Manjulakumari   
Online First Date: 29 December 2017    Issue Date: 10 January 2018
 Cite this article:   
Kavya Krishnappa,Naveen Kumar Mallesh,Srikantaradhya Chidananda Sharma, et al. Midostaurin inhibits hormone-refractory prostate cancer PC-3 cells by modulating nPKCs and AP-1 transcription factors and their target genes involved in cell cycle[J]. Front. Biol., 2017, 12(6): 421-429.
 URL:  
https://academic.hep.com.cn/fib/EN/10.1007/s11515-017-1475-x
https://academic.hep.com.cn/fib/EN/Y2017/V12/I6/421
Gene Primer sequence (5¢®3¢) Annealing temp. (°C) Product size (bp) Reference
nPKC isoform
PKC-δ F: CTGCAAGAAGAACAATGGCAAG
R:ATCCACGTCCTCCAGGAAATACT 		61 97 Present study
PKC-ε F:CTTCTCGACCCCTACATTGCC
R:GCAGGTGCAGACTTGACACTG 		61 449 Present study
AP-1 factors
c-Jun F: GCCTACAGATGAACTCTTTCTGGC
R: CCTGAAACATCGCACTATCCTTTG 		64 525 Babu et al., 2013
Jun-D F: CGCAGCCTCAAACCCTGCCTTTCC
R: AAACAGGAATGTGGACTCGTAG 		64 500 Babu et al., 2013
Jun- B F: CCAGTCCTTCCACCTCGACGTTTACAAG
R: GACTAAGTGCGTGTTTCTTTTCCACAGTAC 		58 257 Babu et al., 2013
c-Fos F: TCTTCCTTCGTCTTCACC
R: AATCAGAACACACTATTGCC 		58 577 Babu et al., 2013
Fra-1 F: AGGAAGGAACTGACCGAC
R: GAAGGGGAGGAGACATTG 		60 497 Babu et al., 2013
Fra-2 F: AGGAGGAGAGATGAGCAG
R: GGATAGGTGAAGACGAGG 		60 518 Babu et al., 2013
Fos-B F: TGTCCCAGGGAAATGTTTCAGGC
R: ACTGGTAGTTCCGCTGGTGGAAGG 		56 451 Present study
Cell cycleregulators and apoptotic genes
P53 F: GAGCCCCCTCTGAGTCAG
R: GCAAAAACATCTTGTTGAG 		56 375 Babu et al., 2013
P21 F:GATCACAAGCAGTGGGGTGA
R:CTGAGTGACTGCACGACCTT 		58 160 Present study
Cyclin D1 F: AGACCTGCGCGCCCTCGGTG
R: GTAGTAGGACAGGAAGTTGTTG 		58 574 Babu et al., 2013
TNF-α F: CAAGCCTGTAGCCCATGTTGTAGC
R: ATCCCAAAGTAGACCTGCCCAGAC 		58 430 Pail et al., 2013
Bax F: AAGCTGAGCGAGTGTCTCAAGCGC
R: TCCCGCCACAAAGATGGTCACG 		61 366 Babu et al., 2013
Bcl-2 F: AGATGTCCAGCCAGCTGCACCTGAC
R: AGATAGGCACCCAGGGTGATGCAAGCT 		62 365 Babu et al., 2013
Caspase-8 F: GATATTGGGGAACAACTGGAC
R: CATGTCATCATCCAGTTTGCA 		58 366 Present Study
β-actin F: TACCACTGGCATCGTGATGGACT
R: TCCTTCTGCATCCTGTCGGCAAT 		62 516 Babu et al., 2013
Tab.1  Sequence of primersused in RT-PCR studies
Fig.1  Effect of midostaurin onthe viability of PC-3 cells.
Fig.2  Effect of PMA or midostaurinor PMA with midostaurin on the mRNAs of novel PKC isozymes in PC-3cells.
Fig.3  Effect of midostaurin onthe PMA-induced expression of mRNAs of AP-1 factors in PC-3 cells.
Fig.4  Effect of midostaurin onmRNAs of AP-1 regulated cell cycle regulators and apoptotic genesin PC-3 cells.
Fig.5  Effect of midostaurin onPMA-induced expression of anti-apoptotic Bcl-2 protein levels in PC-3cells.
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