Involvement of Transient Receptor Potential Melastatin 7 Channels in Sophorae Radix-induced Apoptosis in Cancer Cells - Sophorae Radix and TRPM7 -

  • cc icon
  • ABSTRACT

    Sophorae Radix (SR) plays a role in a number of physiologic and pharmacologic functions in many organs.

    Objective

    The aim of this study was to clarify the potential role for transient receptor potential melastatin 7 (TRPM7) channels in SR-inhibited growth and survival of AGS and MCF-7 cells, the most common human gastric and breast adenocarcinoma cell lines.

    Methods

    The AGS and the MCF-7 cells were treated with varying concentrations of SR. Analyses of the caspase-3 and - 9 activity, the mitochondrial depolarization and the poly (ADPribose) polymerase (PARP) cleavage were conducted to determine if AGS and MCF-7 cell death occured by apoptosis. TRPM7 channel blockers (Gd3+ or 2-APB) and small interfering RNA (siRNA) were used in this study to confirm the role of TRPM7 channels. Furthermore, TRPM7 channels were overexpressed in human embryonic kidney (HEK) 293 cells to identify the role of TRPM7 channels in AGS and MCF-7 cell growth and survival.

    Results

    The addition of SR to a culture medium inhibited AGS and MCF-7 cell growth and survival. Experimental results showed that the caspase-3 and -9 activity, the mitochondrial depolarization, and the degree of PARP cleavage was increased. TRPM7 channel blockade, either by Gd3+ or 2-APB or by suppressing TRPM7 expression with small interfering RNA, blocked the SR-induced inhibition of cell growth and survival. Furthermore, TRPM7 channel overexpression in HEK 293 cells exacerbated SR-induced cell death.

    Conclusions

    These findings indicate that SR inhibits the growth and survival of gastric and breast cancer cells due to a blockade of the TRPM7 channel activity. Therefore, TRPM7 channels may play an important role in the survival of patients with gastric and breast cancer.


  • KEYWORD

    Sophorae Radix , transient receptor potential melastatin 7 (TRPM7) channel , gastric cancer , breast cancer

  • 1. Kim H, Lee MR, Lee GS, An WG, Cho SI 2012 Effect of Sophora flavescens Aiton extract on degranulation of mast cells and contact dermatitis induced by dinitrofluorobenzene in mice. [J Ethnopharmacol.] Vol.142 P.253-8 google
  • 2. Liu T, Song Y, Chen H, Pan S, Sun X 2010 Matrine inhibits proliferation and induces apoptosis of pancreatic cancer cells in vitro and in vivo. [Biol Pharm Bull.] Vol.33 P.1740-5 google
  • 3. Ding Y, Tian RH, Kinjo J, Nohara T, Kitagawa I 1992 Three new oleanene glycosides from Sophora flavescens. [Chem Pharm Bull.] Vol.40 P.2990-4 google
  • 4. Woo ER, Kwak JH, Kim HJ, Park H 1998 A new prenylated flavonol from the roots of Sophora flavescens. [J Nat Prod.] Vol.61 P.1552-4 google
  • 5. Kang TH, Jeong SJ, Ko WG, Kim NY, Lee BH, Inagaki M 2000 Cytotoxic lavandulyl flavanones from Sophora flavescens. [J Nat Prod.] Vol.63 P.680-1 google
  • 6. Son JK, Park JS, Kim JA, Kim Y, Chung SR, Lee SH 2003 Prenylated flavonoids from the roots of Sophora flavescens with tyrosinase inhibitory activity. [Planta Med.] Vol.69 P.559-61 google
  • 7. Kim SJ, Son KH, Chang HW, Kang SS, Kim HP 2003 Tyrosinase inhibitory prenylated flavonoids from Sophora flavescens. [Biol Pharm Bull.] Vol.26 P.1348-50 google
  • 8. Ding P, Chen D, Bastow KF, Nyarko AK, Wang X, Lee KH 2004 Cytotoxic isoprenylated flavonoids from the roots of Sophora flavescens. [Helv Chim Acta.] Vol.87 P.2574-80 google
  • 9. Sato S, Takeo J, Aoyama C, Kawahara H 2007 Na+-glucose cotransporter (SGLT) inhibitory flavonoids from the roots of Sophora flavescens. [Bioorg Med Chem.] Vol.15 P.3445-9 google
  • 10. De Naeyer A, Vanden Berghe W, Pocock V, Milligan S, Haegeman G, De Keukeleire D 2004 Estrogenic and anticarcinogenic properties of kurarinone, a lavandulyl flavanone from the roots of Sophora flavescens. [J Nat Prod.] Vol.67 P.1829-32 google
  • 11. Ryu YB, Curtis-Long MJ, Kim JH, Jeong SH, Yang MK, Lee KW 2008 Pterocarpans and flavanones from Sophora flavescens displaying potent neuraminidase inhibition. [Bioorg Med Chem Lett.] Vol.18 P.6046-9 google
  • 12. Kim BJ, Park EJ, Lee JH, Jeon JH, Kim SJ, So I 2008 Suppression of transient receptor potential melastatin 7 channel induces cell death in gastric cancer. [Cancer Sci.] Vol.99 P.2502-9 google
  • 13. Guilbert A, Gautier M, Dhennin-Duthille I, Haren N, Sevestre H, Ouadid-Ahidouch H 2009 Evidence that TRPM7 is required for breast cancer cell proliferation. [Am J Physiol Cell Physiol.] Vol.297 P.C493-502 google
  • 14. Clapham DE 2003 TRP channels as cellular sensors. [Nature.] Vol.426 P.517-24 google
  • 15. Nadler MJ, Hermosura MC, Inabe K, Perraud AL, Zhu Q, Stokes AJ 2001 LTRPC7 is a Mg·ATP-regulated divalent cation channel required for cell viability. [Nature.] Vol.411 P.590-5 google
  • 16. Runnels LW, Yue L, Clapham DE 2001 TRP-PLIK, a bifunctional protein with kinase and ion channel activities. [Science.] Vol.291 P.1043-7 google
  • 17. Jin J, Desai BN, Navarro B, Donovan A, Andrews NC, Clapham DE 2008 Deletion of Trpm7 disrupts embryonic development and thymopoiesis without altering Mg2+ homeostasis. [Science.] Vol.322 P.756-60 google
  • 18. Jiang J, Li MH, Inoue K, Chu XP, Seeds J, Xiong ZG 2007 Transient receptor potential melastatin 7-like current in human head and neck carcinoma cells: role in cell proliferation. [Cancer Res.] Vol.67 P.10929-38 google
  • 19. Schmitz C, Perraud AL, Johnson CO, Inabe K, Smith MK, Penner R 2003 Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7. [Cell.] Vol.114 P.191-200 google
  • 20. Lang F, Foller M 2005 Lang KS, Lang PA, Ritter M, Gulbins E, et al. Ion channels in cell proliferation and apoptotic cell death. [J Membr Biol.] Vol.205 P.147-57 google
  • 21. Razik MA, Cidlowski JA 2002 Molecular interplay between ion channels and the regulation of apoptosis. [Biol Res.] Vol.35 P.203-7 google
  • 22. Schonherr R. 2005 Clinical relevance of ion channels for diagnosis and therapy of cancer. [J Membr Biol.] Vol.205 P.175-84 google
  • 23. Lehen'kyi V, Shapovalov G, Skryma R, Prevarskaya N 2011 Ion channnels and transporters in cancer. 5. Ion channels in control of cancer and cell apoptosis. [Am J Physiol Cell Physiol.] Vol.301 P.C1281-9 google
  • 24. Kunzelmann K 2005 Ion channels and cancer. [J Membr Biol.] Vol.205 P.159-73 google
  • 25. Abdul M, Hoosein N. 2002 Voltage-gated potassium ion channels in colon cancer. [Oncol Rep.] Vol.9 P.961-4 google
  • 26. Abdul M,, Hoosein N. 2002 Voltage-gated sodium ion channels in prostate cancer: expression and activity. [Anticancer Res.] Vol.22 P.1727-30 google
  • 27. Jirsch J, Deeley RG, Cole SP, Stewart AJ, Fedida D 1993 Inwardly rectifying K+ channels and volume-regulated anion channels in multidrug-resistant small cell lung cancer cells. [Cancer Res.] Vol.53 P.4156-60 google
  • 28. Shuba YM, Prevarskaya N, Lemonnier L, Van Coppenolle F, Kostyuk PG, Mauroy B 2000 Volume-regulated chloride conductance in the LNCaP human prostate cancer cell line. [Am J Physiol Cell Physiol.] Vol.279 P.C1144-1154 google
  • 29. Kim BJ, Nah SY, Jeon JH, So I, Kim SJ 2011 Transient receptor potential melastatin 7 channels are involved in ginsenoside Rg3-induced apoptosis in gastric cancer cells. [Basic Clin Pharmacol Toxicol.] Vol.109 P.233-9 google
  • 30. Owsianik G, D’Hoedt D, Voets T, Nilius B 2006 Structure-function relationship of the TRP channel superfamily. [Rev Physiol Biochem Pharmacol.] Vol.156 P.61-90 google
  • 31. Nilius B, Owsianik G, Voets T, Peters JA 2007 Transient receptor potential cation channels in disease. [Physiol Rev.] Vol.87 P.165-217 google
  • 32. Shapovalov G, Lehen'kyi V, Skryma R, Prevarskaya N 2011 TRP channels in cell survival and cell death in normal and transformed cells. [Cell Calcium.] Vol.50 P.295-302 google
  • 33. Fixemer T, Wissenbach U, Flockerzi V, Bonkhoff H 2003 Expression of the Ca2+-selective cation channel TRPV6 in human prostate cancer: a novel prognostic marker for tumor progression. [Oncogene.] Vol.22 P.7858-61 google
  • 34. Domotor A, Peidl Z, Vincze A, Hunyady B, Szolcsanyi J, Kereskay L 2005 Immunohistochemical distribution of vanilloid receptor, calcitonin-gene related peptide and substance P in gastrointestinal mucosa of patients with different gastrointestinal disorders. [Inflammopharmacology.] Vol.13 P.161-77 google
  • 35. Hartel M, di Mola FF, Selvaggi F, Mascetta G, Wente MN, Felix K 2006 Vanilloids in pancreatic cancer: potential for chemotherapy and pain management. [Gut] Vol.55 P.519-28 google
  • 36. Lazzeri M, Vannucchi MG, Spinelli M, Bizzoco E, Beneforti P, Turini D 2005 Transient receptor potential vanilloid type 1 (TRPV1) expression changes from normal urothelium to transitional cell carcinoma of human bladder. [Eur Urol.] Vol.48 P.691-8 google
  • 37. Sanchez MG, Sanchez AM, Collado B, Malagarie-Cazenave S, Olea N, Carmena MJ 2005 Expression of the transient receptor potential vanilloid 1 (TRPV1) in LNCaP and PC-3 prostate cancer cells and in human prostate tissue. [Eur J Pharmacol.] Vol.515 P.20-7 google
  • 38. Duncan LM, Deeds J, Hunter J, Shao J, Holmgren LM, Woolf EA 1998 Down-regulation of the novel gene melastatin correlates with potential for melanoma metastasis. [Cancer Res.] Vol.58 P.1515-20 google
  • 39. Tsavaler L, Shapero MH, Morkowski S, Laus R. 2001 Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins. [Cancer Res.] Vol.61 P.3760-9 google
  • 40. Heshall SM, Afar DE, Hiller J, Horvath LG, Quinn DI, Rasiah KK 2003 Survival analysis of genome-wide gene expression profiles of prostate cancers identifies new prognostic targets of disease relapse. [Cancer Res.] Vol.63 P.4196-203 google
  • 41. Minke B, CooK B 2002 TRP channel proteins and signal transduction. [Physiol Rev.] Vol.82 P.429-72 google
  • 42. Montell C, Birnbaumer L, Flockerzi V 2002 The TRP channels, a remarkably functional family. [Cell.] Vol.108 P.595-8 google
  • 43. Montell C. 2005 The TRP superfamily of cation channels. [Sci STKE.] Vol.2005 P.re3 google
  • 44. He Y, Yao G, Savoia C, Touyz RM 2005 Transient receptor potential melastatin 7 ion channels regulate magnesium homeostasis in vascular smooth muscle cells: role of angiotensin II. [Circ Res] Vol.96 P.207-15 google
  • 45. Hanano T, Hara Y, Shi J, Morita H, Umebayashi C, Mori E 2004 Involvement of TRPM7 in cell growth as a spontaneously activated Ca2+ entry pathway in human retinoblastoma cells. [J Pharmacol Sci.] Vol.95 P.403-19 google
  • 46. Aarts M, Iihara K, Wei WL, Xiong ZG, Arundine M, Cerwinski W 2003 A key role for TRPM7 channels in anoxic neuronal death. [Cell.] Vol.115 P.863-77 google
  • 47. Krapivinsky G, Mochida S, Krapivinsky L, Cibulsky SM, Clapham DE 2006 The TRPM7 ion channel functions in cholinergic synaptic vesicles and affects transmitter release. [Neuron.] Vol.52 P.485-96 google
  • 48. Su D, May JM, Koury MJ, Asard H 2006 Human erythrocyte membranes contain a cytochrome b561 that may be involved in extracellular ascorbate recycling. [J Biol Chem.] Vol.281 P.39852-9 google
  • 49. Kim BJ, Lim HH, Yang DK, Jun JY, Chang IY, Park CS 2005 Melastatin-type transient receptor potential channel 7 is required for intestinal pacemaking activity. [Gastroenterology.] Vol.129 P.1504-17 google
  • 50. Wykes RC, Lee M, Duffy SM, Yang W, Seward EP, Bradding P 2007 Functional transient receptor potential melastatin 7 channels are critical for human mast cell survival. [J Immunol.] Vol.179 P.4045-52 google
  • 51. Abed E, Moreau R 2007 Importance of melastatin-like transient receptor potential 7 and cations (magnesium, calcium) in human osteoblast-like cell proliferatio [Cell Prolif.] Vol.40 P.849-65 google
  • [Figure 1] Sophorae Radix (SR) induces cell death in AGS and MCF-7 cells. (A) The AGS cells were incubated with SR at the indicated concentrations for 72 hrs prior to MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assays. Distilled water was used as a vehicle. Cell viability is expressed as a value relative to that of the untreated cells which is set to 100%. (B) Time course response to SR. AGS cell viability is expressed as a value relative to that of the cells treated with a vehicle and harvested at zero time which is set to 100%. (C) The MCF-7 cells were incub-ated with SR at the indicated concentrations for 72 hrs prior to MTT assays. Distilled water was used as a vehicle. Cell viability is expressed as a value relative to that of the untr-eated cells which is set to 100%. (D) Time course response to SR. MCF-7 cell viability is expressed as a value relative to that of the cells treated with a vehicle and harvested at zero time which is set to 100%. The figures show mean ± SEM. *P? 0.05, **P? 0.01.
    Sophorae Radix (SR) induces cell death in AGS and MCF-7 cells. (A) The AGS cells were incubated with SR at the indicated concentrations for 72 hrs prior to MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assays. Distilled water was used as a vehicle. Cell viability is expressed as a value relative to that of the untreated cells which is set to 100%. (B) Time course response to SR. AGS cell viability is expressed as a value relative to that of the cells treated with a vehicle and harvested at zero time which is set to 100%. (C) The MCF-7 cells were incub-ated with SR at the indicated concentrations for 72 hrs prior to MTT assays. Distilled water was used as a vehicle. Cell viability is expressed as a value relative to that of the untr-eated cells which is set to 100%. (D) Time course response to SR. MCF-7 cell viability is expressed as a value relative to that of the cells treated with a vehicle and harvested at zero time which is set to 100%. The figures show mean ± SEM. *P? 0.05, **P? 0.01.
  • [Figure 2] SR triggers apoptosis in AGS cells. (A) Mitochondria membrane depolarization is expressed as a value relative to that of untreated cells which is set to 100%. (B) The cells were cultured with SR at the indicated concentrations for 24 hrs prior to caspase assays. Caspase activity from untreated cells is expressed as 100%. Pan-caspase inhibitor zVAD-fmk (zVAD) at 20 μM was used to validate the analytical method employed. (C) A representative western blot of incubated cells with anti-poly (ADP-ribose) polymerase (PARP) antibody. Cells incubated with SR represent increased PARP cleavage. Glyceraldhyde-3- phosphate dehydrogenase (GAPDH) was used as an internal control. The figures show mean ± SEM. *P? 0.05, **P? 0.01.
    SR triggers apoptosis in AGS cells. (A) Mitochondria membrane depolarization is expressed as a value relative to that of untreated cells which is set to 100%. (B) The cells were cultured with SR at the indicated concentrations for 24 hrs prior to caspase assays. Caspase activity from untreated cells is expressed as 100%. Pan-caspase inhibitor zVAD-fmk (zVAD) at 20 μM was used to validate the analytical method employed. (C) A representative western blot of incubated cells with anti-poly (ADP-ribose) polymerase (PARP) antibody. Cells incubated with SR represent increased PARP cleavage. Glyceraldhyde-3- phosphate dehydrogenase (GAPDH) was used as an internal control. The figures show mean ± SEM. *P? 0.05, **P? 0.01.
  • [Figure 3] SR triggers apoptosis in MCF-7 cells. (A) Mitochondria membrane depolarization is expressed as a value relative to that of untreated cells which is set to 100%. (B) The cells were cultured with SR at the indicated concentrations for 24 hrs prior to caspase assays. Caspase activity from untreat-ed cells is expressed as 100%. Pan-caspase inhibitor zVAD-fmk (zVAD) at 20 μM was used to validate the analytical method employed. (C) A representative western blot of incubated cells with anti-poly (ADP-ribose) polymerase (PARP) antibody. Cells incubated with SR represent increased PARP cleavage. Glyceraldhyde-3- phosphate dehydrogenase (GAPDH) was used as an internal control. The figures show mean ± SEM. *P? 0.05, **P? 0.01.
    SR triggers apoptosis in MCF-7 cells. (A) Mitochondria membrane depolarization is expressed as a value relative to that of untreated cells which is set to 100%. (B) The cells were cultured with SR at the indicated concentrations for 24 hrs prior to caspase assays. Caspase activity from untreat-ed cells is expressed as 100%. Pan-caspase inhibitor zVAD-fmk (zVAD) at 20 μM was used to validate the analytical method employed. (C) A representative western blot of incubated cells with anti-poly (ADP-ribose) polymerase (PARP) antibody. Cells incubated with SR represent increased PARP cleavage. Glyceraldhyde-3- phosphate dehydrogenase (GAPDH) was used as an internal control. The figures show mean ± SEM. *P? 0.05, **P? 0.01.
  • [Figure 4] Inhibition of cell death by transient receptor potential melastatin (TRPM7) blockade. (A) MTT assay induced by different treatment as indicated in AGS (a) and MCF-7 (b) cells. Inhibition of TRPM7 channels by Gd3 or 2-APB reduced SR induced apoptosis. (B) Effect of SR on TRPM7-like current in AGS cells. I-V curves (a) and summary bar graph (b) in the absence (■) or presence (●) of SR. (C) Effect of SR on the TRPM7-like current in MCF-7 cells. I-V curves (a) and summary bar graph (b) in the absence (■) or presence (●) of SR. **P ? 0.01.
    Inhibition of cell death by transient receptor potential melastatin (TRPM7) blockade. (A) MTT assay induced by different treatment as indicated in AGS (a) and MCF-7 (b) cells. Inhibition of TRPM7 channels by Gd3  or 2-APB reduced SR induced apoptosis. (B) Effect of SR on TRPM7-like current in AGS cells. I-V curves (a) and summary bar graph (b) in the absence (■) or presence (●) of SR. (C) Effect of SR on the TRPM7-like current in MCF-7 cells. I-V curves (a) and summary bar graph (b) in the absence (■) or presence (●) of SR. **P ? 0.01.
  • [Figure 5] Effects of RNA interference (RNAi) in AGS and MCF-7 cells and the effect of SR on transient receptor potential melastatin 7 (TRPM7) channel overexpression in human embryonic kidney (HEK) cells. (A) AGS cell viability was increased 72 hrs after transfection with TRPM7siRNA and incubation with SR. (B) MCF-7 cell viability was increased 72 hrs after transfection with TRPM7siRNA and incubation with SR. (C) TRPM7 cells were treated or not treated with tetracycline for 1 day. Cells were incubated with SR, followed by MTT assay. **P? 0.01.
    Effects of RNA interference (RNAi) in AGS and MCF-7 cells and the effect of SR on transient receptor potential melastatin 7 (TRPM7) channel overexpression in human embryonic kidney (HEK) cells. (A) AGS cell viability was increased 72 hrs after transfection with TRPM7siRNA and incubation with SR. (B) MCF-7 cell viability was increased 72 hrs after transfection with TRPM7siRNA and incubation with SR. (C) TRPM7 cells were treated or not treated with tetracycline for 1 day. Cells were incubated with SR, followed by MTT assay. **P? 0.01.