Skip to main content
SpringerLink
Log in

Elucidation of a novel phenformin derivative on glucose-deprived stress responses in HT-29 cells

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

Recently, we developed a variety of phenformin derivatives as selective antitumor agents. Based on previous findings, this study evaluated a promising compound, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on the basis of stress responses in the human colon cancer cell line HT-29 under a serum- and glucose-deprived condition. 2-Cl-Phen triggered morphological changes such as shrinkage and plasma membrane disintegration, as well as a decrease in mitochondrial activity and an increase in LDH leakage. To understand intracellular issues relating to 2-Cl-Phen, this study focused on the expression levels of ER stress-inducible genes and several oncogenic genes. Serum and glucose deprivation significantly induced a variety of ER stress-inducible genes, but a 12-h treatment of 2-Cl-Phen down-regulated expression of several ER stress-related genes, with the exception of GADD153. Interestingly, the expression levels of ATF6α, GRP78, MANF, and CRELD2 mRNA were almost completely decreased by 2-Cl-Phen. This study also observed that a 24-h treatment of 2-Cl-Phen attenuated the expression levels of GRP78, GADD153, and c-Myc protein. The decrease in c-Myc protein occurred before the fluctuation of GRP78 protein, while the expression of c-Myc mRNA showed little change with cotreatment of serum and glucose deprivation with 2-Cl-Phen. To further understand the 2-Cl-Phen-induced down-regulation of ATF6-related genes, this study investigated the stability of ATF6α and GRP78 proteins using NanoLuc-tagged constructs. The expression levels of NanoLuc-tagged ATF6α and GRP78 were significantly down-regulated by 2-Cl-Phen in the presence or absence of the translation inhibitor cycloheximide. Taken together, our novel phenformin derivative 2-Cl-Phen has the unique characteristic of diminishing tumor adaptive responses, especially the expression of ATF6-related genes, as well as that of c-Myc protein, in a transcriptional and posttranscriptional manner under a serum- and glucose-deprived condition. Further characterization of cytotoxic mechanisms related to phenformin derivatives may give new insights into developing additional promising anticancer agents.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

ATF6:

Activating transcription factor 6

CRELD2:

Cysteine-rich with EGF-like domains 2

ER:

Endoplasmic reticulum

GADD153:

Growth arrest and DNA damage-inducible protein 153

GRP78:

78 kDa glucose-regulated protein

GRP94:

94 kDa glucose-regulated protein

G3PDH:

Glyceraldehyde 3-phosphate dehydrogenase

MANF:

Mesencephalic astrocyte-derived neurotrophic factor

XBP1:

X-box binding protein 1

References

  1. Gething MJ, Sambrook J (1992) Protein folding in the cell. Nature 355:33–45

    Article  CAS  PubMed  Google Scholar 

  2. Kim I, Xu W, Reed JC (2008) Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov 7:1013–1030

    Article  CAS  PubMed  Google Scholar 

  3. Zhu C, Johansen FE, Prywes R (1997) Interaction of ATF6 and serum response factor. Mol Cell Biol 17:4957–4966

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, Harding HP, Clark SG, Ron D (2002) IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415:92–96

    Article  CAS  PubMed  Google Scholar 

  5. Harding HP, Zhang Y, Ron D (1999) Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397:271–274

    Article  CAS  PubMed  Google Scholar 

  6. Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K (2001) XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107:881–891

    Article  CAS  PubMed  Google Scholar 

  7. Lee AH, Iwakoshi NN, Glimcher LH (2003) XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol Cell Biol 23:7448–7459

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Okada T, Yoshida H, Akazawa R, Negishi M, Mori K (2002) Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response. Biochem J 366:585–594

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Rutkowski DT, Kaufman RJ (2003) All roads lead to ATF4. Dev Cell 4:442–444

    Article  CAS  PubMed  Google Scholar 

  10. Wang M, Ye R, Barron E, Baumeister P, Mao C, Luo S, Fu Y, Luo B, Dubeau L, Hinton DR, Lee AS (2010) Essential role of the unfolded protein response regulator GRP78/BiP in protection from neuronal apoptosis. Cell Death Differ 17:488–498

    Article  CAS  PubMed  Google Scholar 

  11. Oyadomari S, Mori M (2004) Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 11:381–389

    Article  CAS  PubMed  Google Scholar 

  12. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674

    Article  CAS  PubMed  Google Scholar 

  13. Tomida A, Tsuruo T (1999) Drug resistance mediated by cellular stress response to the microenvironment of solid tumors. Anticancer Drug Des 14:169–177

    CAS  PubMed  Google Scholar 

  14. Fu Y, Lee AS (2006) Glucose regulated proteins in cancer progression, drug resistance and immunotherapy. Cancer Biol Ther 5:741–744

    Article  CAS  PubMed  Google Scholar 

  15. Bi M, Naczki C, Koritzinsky M, Fels D, Blais J, Hu N, Harding H, Novoa I, Varia M, Raleigh J, Scheuner D, Kaufman RJ, Bell J, Ron D, Wouters BG, Koumenis C (2005) ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth. EMBO J 24:3470–3481

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hart LS, Cunningham JT, Datta T, Dey S, Tameire F, Lehman SL, Qiu B, Zhang H, Cerniglia G, Bi M, Li Y, Gao Y, Liu H, Li C, Maity A, Thomas-Tikhonenko A, Perl AE, Koong A, Fuchs SY, Diehl JA, Mills IG, Ruggero D, Koumenis C (2012) ER stress-mediated autophagy promotes Myc-dependent transformation and tumor growth. J Clin Invest 122:4621–4634

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Pyrko P, Schonthal AH, Hofman FM, Chen TC, Lee AS (2007) The unfolded protein response regulator GRP78/BiP as a novel target for increasing chemosensitivity in malignant gliomas. Cancer Res 67:9809–9816

    Article  CAS  PubMed  Google Scholar 

  18. Ri M, Tashiro E, Oikawa D, Shinjo S, Tokuda M, Yokouchi Y, Narita T, Masaki A, Ito A, Ding J, Kusumoto S, Ishida T, Komatsu H, Shiotsu Y, Ueda R, Iwawaki T, Imoto M, Iida S (2012) Identification of toyocamycin, an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER stress-induced XBP1 mRNA splicing. Blood Cancer J 2:e79

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Narise K, Okuda K, Enomoto Y, Hirayama T, Nagasawa H (2014) Optimization of biguanide derivatives as selective antitumor agents blocking adaptive stress responses in the tumor microenvironment. Drug Des Dev Ther 8:701–717

    CAS  Google Scholar 

  20. Oh-hashi K, Tanaka K, Koga H, Hirata Y, Kiuchi K (2012) Intracellular trafficking and secretion of mouse mesencephalic astrocyte-derived neurotrophic factor. Mol Cell Biochem 363:35–41

    Article  CAS  PubMed  Google Scholar 

  21. Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F, Encell LP, Wood KV (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7:1848–1857

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Petrova P, Raibekas A, Pevsner J, Vigo N, Anafi M, Moore MK, Peaire AE, Shridhar V, Smith DI, Kelly J, Durocher Y, Commissiong JW (2003) MANF: a new mesencephalic, astrocyte-derived neurotrophic factor with selectivity for dopaminergic neurons. J Mol Neurosci 20:173–188

    Article  CAS  PubMed  Google Scholar 

  23. Oh-hashi K, Koga H, Ikeda S, Shimada K, Hirata Y, Kiuchi K (2009) CRELD2 is a novel endoplasmic reticulum stress-inducible gene. Biochem Biophys Res Commun 387:504–510

    Article  CAS  PubMed  Google Scholar 

  24. Sircar K, Huang H, Hu L, Cogdell D, Dhillon J, Tzelepi V, Efstathiou E, Koumakpayi IH, Saad F, Luo D, Bismar TA, Aparicio A, Troncoso P, Navone N, Zhang W (2012) Integrative molecular profiling reveals asparagine synthetase is a target in castration-resistant prostate cancer. Am J Pathol 180:895–903

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. van Riggelen J, Yetil A, Felsher DW (2010) MYC as a regulator of ribosome biogenesis and protein synthesis. Nat Rev Cancer 10:301–309

    Article  PubMed  Google Scholar 

  26. Babcock JT, Nguyen HB, He Y, Hendricks JW, Wek RC, Quilliam LA (2013) Mammalian target of rapamycin complex 1 (mTORC1) enhances bortezomib-induced death in tuberous sclerosis complex (TSC)-null cells by a c-MYC-dependent induction of the unfolded protein response. J Biol Chem 288:15687–15698

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Amundson SA, Zhan Q, Penn LZ, Fornace AJ Jr (1998) Myc suppresses induction of the growth arrest genes gadd34, gadd45, and gadd153 by DNA-damaging agents. Oncogene 17:2149–2154

    Article  CAS  PubMed  Google Scholar 

  28. David CJ, Chen M, Assanah M, Canoll P, Manley JL (2010) HnRNP proteins controlled by c-Myc deregulate pyruvate kinase mRNA splicing in cancer. Nature 463:364–368

    Article  CAS  PubMed  Google Scholar 

  29. Kern J, Untergasser G, Zenzmaier C, Sarg B, Gastl G, Gunsilius E, Steurer M (2009) GRP-78 secreted by tumor cells blocks the antiangiogenic activity of bortezomib. Blood 114:3960–3967

    Article  CAS  PubMed  Google Scholar 

  30. Li Z, Wang Y, Wu H, Zhang L, Yang P, Li Z (2014) GRP78 enhances the glutamine metabolism to support cell survival from glucose deficiency by modulating the β-catenin signaling. Oncotarget 5:5369–5380

    Article  PubMed  PubMed Central  Google Scholar 

  31. Harding HP, Zyryanova AF, Ron D (2012) Uncoupling proteostasis and development in vitro with a small molecule inhibitor of the pancreatic endoplasmic reticulum kinase. PERK J Biol Chem 287:44338–44344

    Article  CAS  PubMed  Google Scholar 

  32. Namba T, Ishihara T, Tanaka K, Hoshino T, Mizushima T (2007) Transcriptional activation of ATF6 by endoplasmic reticulum stressors. Biochem Biophys Res Commun 355:543–548

    Article  CAS  PubMed  Google Scholar 

  33. Ron D, Harding H (2007) eIF2α phosphorylation in cellular stress responses and disease. In: Sonenberg N, Hershey J, Mathews M (eds) Translational control., Cold Spring Harbor Monograph SeriesCold Spring Harbor Lab Press, Cold Spring Harbor, New York, pp 345–368

    Google Scholar 

  34. Nadanaka S, Okada T, Yoshida H, Mori K (2007) Role of disulfide bridges formed in the luminal domain of ATF6 in sensing endoplasmic reticulum stress. Mol Cell Biol 27:1027–1043

    Article  CAS  PubMed  Google Scholar 

  35. Higa A, Taouji S, Lhomond S, Jensen D, Fernandez-Zapico ME, Simpson JC, Pasquet JM, Schekman R, Chevet E (2014) Endoplasmic reticulum stress-activated transcription factor ATF6α requires the disulfide isomerase PDIA5 to modulate chemoresistance. Mol Cell Biol 34:1839–1849

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lee CY, Lee MG, Choi KC, Kang HM, Chang YS (2012) Clinical significance of GADD153 expression in stage I non-small cell lung cancer. Oncol Lett 4:408–412

    PubMed  PubMed Central  Google Scholar 

  37. Thevenot PT, Sierra RA, Raber PL, Al-Khami AA, Trillo-Tinoco J, Zarreii P, Ochoa AC, Cui Y, Del Valle L, Rodriguez PC (2014) The stress-response sensor chop regulates the function and accumulation of myeloid-derived suppressor cells in tumors. Immunity 41:389–401

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ye J, Kumanova M, Hart LS, Sloane K, Zhang H, De Panis DN, Bobrovnikova-Marjon E, Diehl JA, Ron D, Koumenis C (2010) The GCN2-ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation. EMBO J 29:2082–2096

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Raykhel I, Alanen H, Salo K, Jurvansuu J, Nguyen VD, Latva-Ranta M, Ruddock L (2007) A molecular specificity code for the three mammalian KDEL receptors. J Cell Biol 179:1193–1204

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Wasylishen AR, Chan-Seng-Yue M, Bros C, Dingar D, Tu WB, Kalkat M, Chan PK, Mullen PJ, Huang L, Meyer N, Raught B, Boutros PC, Penn LZ (2013) MYC phosphorylation at novel regulatory regions suppresses transforming activity. Cancer Res 73:6504–6515

    Article  CAS  PubMed  Google Scholar 

  41. Popov N, Wanzel M, Madiredjo M, Zhang D, Beijersbergen R, Bernards R, Moll R, Elledge SJ, Eilers M (2007) The ubiquitin-specific protease USP28 is required for MYC stability. Nat Cell Biol 9:765–774

    Article  CAS  PubMed  Google Scholar 

  42. Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, Wu M, Ventre J, Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear LJ, Moller DE (2001) Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 108:1167–1174

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kalender A, Selvaraj A, Kim SY, Gulati P, Brûlé S, Viollet B, Kemp BE, Bardeesy N, Dennis P, Schlager JJ, Marette A, Kozma SC, Thomas G (2010) Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPase-dependent manner. Cell Metab 11:390–401

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kickstein E, Krauss S, Thornhill P, Rutschow D, Zeller R, Sharkey J, Williamson R, Fuchs M, Köhler A, Glossmann H, Schneider R, Sutherland C, Schweiger S (2010) Biguanide metformin acts on tau phosphorylation via mTOR/protein phosphatase 2A (PP2A) signaling. Proc Natl Acad Sci USA 107:21830–21835

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Lee J, Chan SL, Lu C, Lane MA, Mattson MP (2002) Phenformin suppresses calcium responses to glutamate and protects hippocampal neurons against excitotoxicity. Exp Neurol 175:161–167

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work is partly supported by the Koshiyama Science and Technology Foundation (to Kensuke Okuda) and the OGAWA Science and Technology Foundation (to Kentaro Oh-hashi).

Author information

Authors and Affiliations

  1. Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Kentaro Oh-hashi, Nao Irie, Yoko Hirata & Kazutoshi Kiuchi

  2. United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Kentaro Oh-hashi, Yoko Hirata & Kazutoshi Kiuchi

  3. Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Pharmaceutical University, 1-25-4 Daigakunishi, Gifu, Japan

    Takayuki Sakai & Hideko Nagasawa

  4. Laboratory of Bioorganic and Natural Products Chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyama-kita, Higashinada, Kobe, 658-8558, Japan

    Kensuke Okuda

Authors
  1. Kentaro Oh-hashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nao Irie
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Takayuki Sakai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kensuke Okuda
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hideko Nagasawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yoko Hirata
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kazutoshi Kiuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kentaro Oh-hashi.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11010_2016_2747_MOESM1_ESM.ppt

Supplementary material 1 (PPT 10203 kb) Supplementary Fig. 1 Effect of 2-Cl-Phen treatment on proliferation and damage in HT-29 cells cultured with the serum-containing or serum- and glucose-deprived medium. HT-29 cells in the serum-containing or serum- and glucose-deprived medium were treated with 2-Cl-Phen (50 μM) or vehicle for the indicated days. The arrows indicate the typical damaged cells

11010_2016_2747_MOESM2_ESM.pptx

Supplementary material 2 (PPTX 8425 kb) Supplementary Fig. 2 Expression of EGFP and NanoLuc-tagged ATF6α in COS7 cells A) The schematic structures of EGFP and NanoLuc-tagged ATF6α used in this study. Each construct has a Flag epitope (F) at the N-terminus. EGFP or NanoLuc (NL) gene was inserted between Flag tag and the N-terminus of full-length (FL) or nuclear localized (N) ATF6α. B) Forty-eight hours after transfection of the indicated construct into COS7 cells in a 6-well plate, fluorescent images were obtained by fluorescent microscopy (OLYMPUS). The scale bar is 100 μm. C) Forty-eight hours after transfection of the indicated construct into COS7 cells in a 6-well plate, the cells were lysed, and NanoLuc activity was measured using an aliquot of the lysate as described in the Materials and Methods section

11010_2016_2747_MOESM3_ESM.pptx

Supplementary material 3 (PPTX 8575 kb) Supplementary Fig. 3 Expression of EGFP and NanoLuc-tagged GRP78 in COS7 cells A) The schematic structures of EGFP and NanoLuc (NL)-tagged GRP78 (SP-EGFP-GRP78 and SP-NL-GRP78) used in this study. SP indicates a signal peptide sequence at the N-terminus. B) Forty-eight hours after transfection of the indicated construct into COS7 cells in a 6-well plate, fluorescent images were obtained by fluorescent microscopy. The scale bar is 100 μm. C) Forty-eight hours after transfection of the indicated construct into COS7 cells in a 6-well plate, the cells were lysed, and NanoLuc activity was measured using an aliquot of the lysate as described in the Materials and Methods section

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Oh-hashi, K., Irie, N., Sakai, T. et al. Elucidation of a novel phenformin derivative on glucose-deprived stress responses in HT-29 cells. Mol Cell Biochem 419, 29–40 (2016). https://doi.org/10.1007/s11010-016-2747-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-016-2747-5

Keywords

Search

Navigation