Advertisement

Apoptosis

, Volume 22, Issue 11, pp 1362–1379 | Cite as

Modulation of adenylate cyclase signaling in association with MKK3/6 stabilization under combination of SAC and berberine to reduce HepG2 cell survivability

  • Dipanwita Sengupta
  • Kaustav Dutta Chowdhury
  • Sujan Chatterjee
  • Avik Sarkar
  • Soumosish Paul
  • Pradip Kumar Sur
  • Gobinda Chandra Sadhukhan
Original Paper

Abstract

Cancer cells often have faulty apoptotic pathways resulting in sustenance of survivability, tumour metastasis and resistance to anticancer drugs. Alternate strategies are sought to improve therapeutic efficacy and therefore HepG2 cells were treated with S-allyl-cysteine (SAC) and berberine (BER) to analyze their mechanistic impact upon necroptosis along with its interacting relationship to apoptosis. In the present study we observed that SAC and BER exposure reduced NFκβ nuclear translocation through adenylate cyclase-cAMP-protein kinaseA axis and eventually evaded c-FLIP inhibition. Effective RIP1 k63-polyubiquitination and persistent MKK3/MKK6 expression during drug treatment potentiated caspase8 activity via p53—DISC conformation. Resultant tBid associated lysosomal protease mediated AIF truncation induced DNA fragmentation and persuaded effector caspase mediated scramblase activation resulting induction of necroptosis in parallel to apoptotic events. SAC+BER effectively reduced Rb-phosphorylation resulting insignificant nuclear E2F presence led to ending of cell proliferation. Therefore necroptosis augmented the drug response and may be targeted alongside cell proliferation inhibition in formation of efficient therapeutics against liver cancer.

Keywords

Adenylate cyclase CathepsinB Rb phosphorylation RIP1-K63-polyubiquitination tAIF 

Abbreviations

DISC

Death-inducing signaling complex

AIF

Apoptosis-inducing factor

SAC

S-allyl-cysteine

BER

Berberine

LMP

Lysosomal membrane potential

MMP

Mitochondrial membrane potential

TNFα

Tumor necrosis factor alpha

TNFR1

Tumor necrosis factor receptor 1

WST-1

Water soluble tetrazolium salts-1

ELISA

Enzyme linked immune-sorbent assay

ATF2

Association of activating transcription factor 2

TAK1

Transforming growth factor β activated kinase 1

PKA

Protein kinase A

CKI

Cyclin dependent kinase inhibitor

CDK

Cyclin dependent kinase

EMSA

Enzyme mobility shift assay

PS

Phosphatidyl serine

GFP

Green fluorescent protein

AO

Acridine Orange

FACS

Fluorescence-activated cell sorting

MPT

Mitochondrial permeability transition pore

APLT

Amino phospholipid translocase

FLIP

Flice inhibitory protein

Notes

Acknowledgements

This work was supported by the grant from University Grants Commission (F.PSW-85/13-14 (ERO) dated 18.03.2014). Authors are grateful to N. S. Roy and U. Bhattacharya for critical reading, scientific discussions and helpful suggestions, S. Roy for statistical support and analysis of the data, D. Bhowmick, CRNN, University of Calcutta for flow cytometric and confocal microscopic analysis.

Compliance with ethical standards

Conflict of interest

The authors declared that they have no conflict of interest.

Supplementary material

10495_2017_1407_MOESM1_ESM.doc (74 kb)
Supplementary material 1 (DOC 73 KB)
10495_2017_1407_MOESM2_ESM.tif (3.6 mb)
Supplementary material 2 (TIF 3678 KB)

References

  1. 1.
    Lee J, Seong J (2012) The optimal selection of radiotherapy treatment for hepatocellular carcinoma. Gut Liver 6:139–148. doi: 10.5009/gnl.2012.6.2.139 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Livraghi T, Solbiati L, Meloni MF et al (2003) Treatment of focal liver tumors with percutaneous radio-frequency ablation: complications encountered in a multicenter study. Radiology 226:441–451. doi: 10.1148/radiol.2262012198 CrossRefPubMedGoogle Scholar
  3. 3.
    Dhanasekaran R, Limaye A, Cabrera R (2012) Hepatocellular carcinoma: current trends in worldwide epidemiology, risk factors, diagnosis, and therapeutics. Hepat Med 4:19–37. doi: 10.2147/HMER.S16316 PubMedPubMedCentralGoogle Scholar
  4. 4.
    Sengupta D, Dutta Chowdhury K, Sarkar A et al (2014) Berberine and S-allyl-cysteine mediated amelioration of DEN + CCl4 induced hepatocarcinoma. Biochim Biophys Acta 1840:219–244. doi: 10.1016/j.bbagen.2013.08.020 CrossRefPubMedGoogle Scholar
  5. 5.
    Marques-Fernandez F, Planells-Ferrer L, Gozzelino R et al (2013) TNFα induces survival through the FLIP-L-dependent activation of the MAPK/ERK pathway. Cell Death Dis 14(4):e493. doi: 10.1038/cddis.2013.25 CrossRefGoogle Scholar
  6. 6.
    Fan C, Yang J, Engelhardt JF (2002) Temporal pattern of NFκB activation influences apoptotic cell fate in a stimuli-dependent fashion. J Cell Sci 115:4843–4853. doi: 10.1242/jcs.00151 CrossRefPubMedGoogle Scholar
  7. 7.
    Loeffler M, Daugas E, Susin SA et.al (2001) Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor. FASEB J 15:758–767. doi: 10.1096/fj.00-0388com CrossRefPubMedGoogle Scholar
  8. 8.
    Zhuang R, Zhang Y, Zhang R et.al (2008) Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography. Protein Expr Purif 59:138–143. doi: 10.1016/j.pep.2008.01.020 CrossRefPubMedGoogle Scholar
  9. 9.
    Chaturvedi MM, Mukhopadhyay A, Aggarwal BB (2000) Assay for redox-sensitive transcription factor. Methods Enzymol 319:585–602. doi: 10.1016/S0076-6879(00)19055-X CrossRefPubMedGoogle Scholar
  10. 10.
    Guo K, Kang NX, Li Y (2009) Regulation of HSP27 on NF-κB pathway activation may be involved in metastatic hepatocellular carcinoma cells apoptosis. BMC Cancer 9:100–109. doi: 10.1186/1471-2407-9-100 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Chen KF, Yeh PY, Hsu C et al (2009) Bortezomib overcomes tumour necrosis factor-related apoptosis-inducing ligand resistance in hepatocellular carcinoma cells in part through the inhibition of the phosphatidylinositol 3-kinase/akt pathway. J Biol Chem 284:11121–11133. doi: 10.1074/jbc.M806268200 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Blink BVD, Juffermans NP, ten Hove T et al. (2001) p38 Mitogen-activated protein kinase inhibition increases cytokine release by macrophages in vitro and during infection in vivo. J Immunol 166:582–587. doi: 10.4049/jimmunol.166.1.582 CrossRefPubMedGoogle Scholar
  13. 13.
    Kim SY, Baik KH, Baek KH et al (2014) S6K1 negatively regulates TAK1 activity in the toll-like receptor signaling pathway. Mol Cell Biol 34:510–521. doi: 10.1128/MCB.01225-13 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Kwon M, Fernández JR, Zegarek GF et al (2011) BDNF-promoted increases in proximal dendrites occur via CREB-dependent transcriptional regulation of cypin. J Neurosci 31:9735–9745. doi: 10.1523/JNEUROSCI.6785-10.2011 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Penas C, Ramachandran V, Simanski S, Lee C et al (2014) Casein kinase 1δ-dependent Wee1 protein degradation. J Biol Chem 289:18893–18903. doi: 10.1074/jbc.M114.547661 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Rundhaug JE, Fischer SM (2010) Molecular mechanisms of mouse skin tumor promotion. Cancers (Basel) 2:436–482. doi: 10.3390/cancers2020436 CrossRefGoogle Scholar
  17. 17.
    Jimeno A, Feldmann G, Suárez-Gauthier A et al (2009) A direct pancreatic cancer xenograft model as a platform for cancer stem cell therapeutic development. Mol Cancer Ther 8:310–314. doi: 10.1158/1535-7163.MCT-08-0924 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Habelhah H (2010) Emerging complexity of protein ubiquitination in the NF-κβ pathway. Genes Cancer 1:735–747. doi: 10.1177/1947601910382900 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Li S, Wang L, Dorf ME (2009) PKC phosphorylation of TRAF2 mediates IKKα/β recruitment and K63-linked polyubiquitination. Mol Cell 33:30–42. doi: 10.1016/j.molcel.2008.11.023 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Scholz R, Sidler CL, Thali RL et al (2010) Autoactivation of transforming growth factor β-activated kinase1 is a sequential bimolecular process. J Biol Chem 285:25753–25766. doi: 10.1074/jbc.M109.093468 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Parrish AB, Freel CD, Kornbluth S (2013) Cellular mechanisms controlling caspase activation and function. Cold Spring Harb Perspect Biol 5: a008672. doi: 10.1101/cshperspect.a008672 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Witt J, Barisic S, Schumann E, et al (2009) Mechanism of PP2A-mediated IKKβ dephosphorylation: a systems biological approach. BMC Syst Biol. 3:71. doi: 10.1186/1752-0509-3-71 PubMedPubMedCentralGoogle Scholar
  23. 23.
    Kantari C, Walczak H (2011) Caspase-8 and bid: caught in the act between death receptors and mitochondria. Biochim Biophys Acta 1813:558–563. doi: 10.1016/j.bbamcr.2011.01.026 CrossRefPubMedGoogle Scholar
  24. 24.
    Bhattacharyya J, Thompson K, Sayeed MM (1991) Calcium-dependent and calcium-independent protease activities in skeletal muscle during sepsis. Circ Shock 35:117–122. doi: 10.1172/JCI117588 PubMedGoogle Scholar
  25. 25.
    Smrz D, Lebduska P, Draberova L et al (2008) Engagement of phospholipid scramblase1 in activated cells: implication for phosphatidylserine externalization and exocytosis. J Biol Chem 283(16):10904–10918. doi: 10.1074/jbc.M710386200 CrossRefPubMedGoogle Scholar
  26. 26.
    Delavallee L, Cabon L, Galan-Malo P et al (2011) AIF-mediated caspase-independent necroptosis: a new chance for targeted therapeutics. IUBMB Life 63(4):221–232. doi: 10.1002/iub.432 CrossRefPubMedGoogle Scholar
  27. 27.
    Baritaud M, Cabon L, Delavallée L et al (2012) AIF-mediated caspase-independent necroptosis requires ATM and DNA-PK-induced histone H2AX Ser139 phosphorylation. Cell Death Dis 3:e390. doi: 10.1038/cddis.2012.120 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Wang Y, Wang H, Tao Y, Zhang S et al (2014) Necroptosis inhibitor necrostatin-1 promotes cell protection and physiological function in traumatic spinal cord injury. Neuroscience 266:91–101. doi: 10.1016/j.neuroscience.2014.02.007 CrossRefPubMedGoogle Scholar
  29. 29.
    Karamitopoulou E, Cioccari L, Jakob S et al (2007) Active caspase 3 and DNA fragmentation as markers for apoptotic cell death in primary and metastatic liver tumours. Pathology 39:558–564. doi: 10.1080/00313020701684375 CrossRefPubMedGoogle Scholar
  30. 30.
    Sever-Chroneos Z, Angus SP, Fribourg AF et al (2001) Retinoblastoma tumor suppressor protein signals through inhibition of cyclin-dependent kinase2 activity to disrupt PCNA function in S Phase. Mol Cell Biol 21:4032–4045. doi: 10.1128/MCB.21.12.4032-4045.2001 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Dipanwita Sengupta
    • 1
  • Kaustav Dutta Chowdhury
    • 1
    • 2
  • Sujan Chatterjee
    • 1
  • Avik Sarkar
    • 1
  • Soumosish Paul
    • 1
  • Pradip Kumar Sur
    • 3
  • Gobinda Chandra Sadhukhan
    • 1
  1. 1.Molecular Biology and Tissue Culture Laboratory, Post Graduate Department of ZoologyVidyasagar CollegeKolkataIndia
  2. 2.Cyto-genetics Laboratory, Department of ZoologyRammohon CollegeKolkataIndia
  3. 3.Cyto-genetics Laboratory, Department of ZoologyKanchrapara CollegeKanchraparaIndia

Personalised recommendations