Caffeic Acid Induces Intrinsic Apoptotic Pathway in MG-63 Osteosarcoma Cells Through Bid Truncation and Cytochrome c Release

Ferry Sandra, Muhammad Ihsan Rizal, Ayasha Hajjar Audreyandra Wahid, Monica Andajana, Maria Celinna


BACKGROUND: Caffeic acid has been reported to induce apoptosis in MG-63 osteosarcoma cells via caspases activation. However, apoptotic pathway that is involved in the caffeic acid-induced apoptosis is still unclear. Present study aimed to investigate the role of cytochrome c (Cyt c) release and BH3-interacting death (Bid) activation in caffeic acid-induced apoptosis in MG-63 osteosarcoma cells.

METHODS: MG-63 cells were cultured, pretreated with/without Z-VAD FMK and treated with/without 10 μg/mL caffeic acid. Treated MG-63 cells were then lysed, homogenized, and processed further to prepare cell lysate and mitochondrial fraction. Immunoblotting method was used to measure the amount of Bid and truncated Bid (t-Bid) as well as mitochondrial and cytosolic Cyt c.

RESULTS: The amount of Bid and mitochondrial Cyt c in MG-63 cells decreased in a time-dependent manner, while the amount of t-Bid and cytosolic Cyt c increased in a time-dependent manner. By pretreatment of 100 μM Z-VAD-FMK for 2 h, the amount of Bid and mitochondrial Cyt c was significantly higher, while the amount of t-Bid and cytosolic Cyt c was significantly lower after caffeic acid treatment for 6 and 12 h compared to MG-63 cells that were not pretreated.

CONCLUSION: Caffeic acid could induce Cyt c release through the activation of Bid in MG-63 osteosarcoma cells.

KEYWORDS: caffeic acid, osteosarcoma, MG-63 cells, Bid, t-Bid, cytochrome c, Z-VAD-FMK

Full Text:



Klein MJ, Siegal GP. Osteosarcoma: Anatomic and histologic variants. Am J Clin Pathol. 2006; 125(4): 555–81, CrossRef.

Baumhoer D, Brunner P, Eppenberger-Castori S, Smida J, Nathrath M, Jundt G. Osteosarcomas of the jaws differ from their peripheral counterparts and require a distinct treatment approach. Experiences from the DOESAK Registry. Oral Oncol. 2014; 50(2): 147–53, CrossRef.

Ta HT, Dass CR, Choong PFM, Dunstan DE. Osteosarcoma treatment: State of the art. Cancer Metastasis Rev. 2009; 28(1–2): 247–63, CrossRef.

Meyers PA, Schwartz CL, Krailo M, Kleinerman ES, Betcher D, Bernstein ML, et al. Osteosarcoma: A randomized, prospective trial of the addition of ifosfamide and/or muramyl tripeptide to cisplatin, doxorubicin, and high-dose methotrexate. J Clin Oncol. 2005; 23(9): 2004–11, CrossRef.

Ferrari S, Meazza C, Palmerini E, Tamburini A, Fagioli F, Cozza R, et al. Nonmetastatic osteosarcoma of the extremity. Neoadjuvant chemotherapy with methotrexate, cisplatin, doxorubicin and ifosfamide. An Italian Sarcoma Group study (ISG/OS-Oss). Tumori. 2014; 100(6): 612–9, CrossRef.

Rahmawati DY, Dwifulqi H, Sandra F. Origin, stemness, marker and signaling pathway of oral cancer stem cell. Mol Cell Biomed Sci. 2020; 4(3): 100–4, CrossRef.

Anderson ME. Update on survival in osteosarcoma. Orthop Clin North Am. 2016; 47(1): 283–92, CrossRef.

Shaikh A, Li F, Li M, He B, He X, Chen G, et al. Present advances and future perspectives of molecular targeted therapy for osteosarcoma. Int J Mol Sci. 2016; 17(4): 506, CrossRef.

Mattila P, Pihlava J, Hellström J. Contents of phenolic acids, alkyl- and alkenylresorcinols, and avenanthramides in commercial grain products. J Agric Food Chem. 2005; 53(21): 8290–5, CrossRef.

Sellappan S, Akoh CC, Krewer G. Phenolic compounds and antioxidant capacity of Georgia-grown blueberries and blackberries. J Agric Food Chem. 2002; 50(8): 2432–8, CrossRef.

Tang QY, Kukita T, Ushijima Y, Kukita A, Nagata K, Sandra F, et al. Regulation of osteoclastogenesis by Simon extracts composed of caffeic acid and related compounds: Successful suppression of bone destruction accompanied with adjuvant-induced arthritis in rats. Histochem Cell Biol. 2006; 125(3): 215–25, CrossRef.

Luís Â, Silva F, Sousa S, Duarte AP, Domingues F. Antistaphylococcal and biofilm inhibitory activities of gallic, caffeic, and chlorogenic acids. Biofouling. 2014; 30(1): 69–79, CrossRef.

Kumaran KS, Prince PSM. Caffeic acid protects rat heart mitochondria against isoproterenol-induced oxidative damage. Cell Stress Chaperones. 2010; 15(6): 791–806, CrossRef.

da Cunha FM, Duma D, Assreuy J, Buzzi FC, Niero R, Campos MM, et al. Caffeic acid derivatives: In vitro and in vivo anti-inflammatory properties. Free Radic Res. 2004; 38(11): 1241–53, CrossRef.

Sandra F, Kukita T, Tang QY, Iijima T. Caffeic acid inhibits NFκB activation of osteoclastogenesis signaling pathway. Indones Biomed J. 2011; 3(3): 216–22, CrossRef.

Sandra F, Kukita T, Muta T, Iijima T. Caffeic acid inhibited receptor activator of nuclear factor κB ligand (RANKL)-tumor necrosis factor (TNF) α-TNF receptor associated factor (TRAF) 6 induced osteoclastogenesis pathway. Indones Biomed J. 2013; 5(3): 173–8, CrossRef.

Sandra F, Briskila J, Ketherin K. RANKL and TNF-α-induced JNK/SAPK osteoclastogenic signaling pathway was inhibited by caffeic acid in RAW-D cells. Indones J Cancer Chemoprevent. 2018; 9(2): 63–7, CrossRef.

Sandra F, Ketherin K. Caffeic acid Inhibits RANKL and TNF-α-induced phosphorylation of p38 mitogen-activated protein kinase in RAW-D cells. Indones Biomed J. 2018; 10(2): 140–3, CrossRef.

Sandra F, Putri J, Limen H, Sarizta B. Caffeic acid inhibits RANKL and TNFα-induced osteoclastogenesis by targeting TAK1-p44/42 MAPK. Indones Biomed J. 2021; 13(4): 433–7, CrossRef.

Xia Y, Shen S, Verma IM. NF-κB, an active player in human cancers. Cancer Immunol Res. 2014; 2(9): 823–30, CrossRef.

Sandra F, Sidharta MA. Caffeic acid induced apoptosis in MG63 osteosarcoma cells through activation of caspases. Mol Cell Biomed Sci. 2017; 1(1): 28–33, CrossRef.

Sandra F, Hudono KF, Putri AA, Putri CAP. Caspase inhibitor diminishes caffeic acid-induced apoptosis in osteosarcoma cells. Indones Biomed J. 2017; 9(3): 160–4, CrossRef.

Fulda S, Debatin KM. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene. 2006; 25(34): 4798–811, CrossRef.

Sandra F, Hendarmin L, Nakao Y, Nakamura N, Nakamura S. Inhibition of Akt and MAPK pathways elevated potential of TNFα in inducing apoptosis in ameloblastoma. Oral Oncol. 2006; 42(1): 38–44, CrossRef.

Sandra F, Matsuda M, Yoshida H, Hirata M. Inositol hexakisphosphate blocks tumor cell growth by activating apoptotic machinery as well as by inhibiting the Akt/NFκB-mediated cell survival pathway. Carcinogenesis. 2002; 23(12): 2031–41, CrossRef.

Lin CL, Chen RF, Chen JYF, Chu YC, Wang HM, Chou HL, et al. Protective effect of caffeic acid on paclitaxel induced anti-proliferation and apoptosis of lung cancer cells involves NF-κB pathway. Int J Mol Sci. 2012; 13(5): 6236–45, CrossRef.

Kim EY, Ryu JH, Kim AK. CAPE promotes TRAIL-induced apoptosis through the upregulation of TRAIL receptors via activation of p38 and suppression of JNK in SK-Hep1 hepatocellular carcinoma cells. Int J Oncol. 2013; 43(4): 1291–300, CrossRef.

Sandra F, Hendarmin L, Nakao Y, Nakamura N, Nakamura S. TRAIL cleaves caspase-8, -9 and -3 of AM-1 cells: A possible pathway for TRAIL to induce apoptosis in ameloblastoma. Tumor Biol. 2005; 26(5): 258–64, CrossRef.

Lee YJ, Kuo HC, Chu CY, Wang CJ, Lin WC, Tseng TH. Involvement of tumor suppressor protein p53 and p38 MAPK in caffeic acid phenethyl ester-induced apoptosis of C6 glioma cells. Biochem Pharmacol. 2003; 66(12): 2281–9, CrossRef.

Chang WC, Hsieh CH, Hsiao MW, Lin WC, Hung YC, Ye JC. Caffeic acid induces apoptosis in human cervical cancer cells through the mitochondrial pathway. Taiwan J Obstet Gynecol. 2010; 49(4): 419–24, CrossRef.

Zhang Y, Yu P, Gao Z, Yuan J, Zhang Z. Caffeic acid n-butyl ester-triggered necrosis-like cell death in lung cancer cell line A549 is prompted by ROS mediated alterations in mitochondrial membrane potential. Eur Rev Med Pharmacol Sci. 2017; 21(7): 1665–71, PMID.

Ito Y, Pandey P, Sporn MB, Datta R, Kharbanda S, Kufe D. The novel triterpenoid CDDO induces apoptosis and differentiation of human osteosarcoma cells by a caspase-8 dependent mechanism. Mol Pharmacol. 2001; 59(5): 1094–9, CrossRef.

Kwon HY, Kim K, An H, Moon H, Kim H, Lee Y. Triptolide induces apoptosis through extrinsic and intrinsic pathways in human osteosarcoma U2OS cells. Indian J Biochem Biophys. 2013; 50(6): 485-91, PMID.

Tao LJ, Zhou XD, Shen CC, Liang CZ, Liu B, Tao Y, et al. Tetrandrine induces apoptosis and triggers a caspase cascade in U2-OS and MG-63 cells through the intrinsic and extrinsic pathways. Mol Med Rep. 2014; 9(1): 345–9, CrossRef.

Tait S, Green D. Caspase-independent cell death: Leaving the set without the final cut. Oncogene. 2008; 27(50): 6452–61, CrossRef.

Kontny U, Lissat A. Apoptosis and drug resistance in malignant bone tumors. In: Heymann D, editor. Bone Cancer: Primary Bone Cancers and Bone Metastases. 2nd ed. Amsterdam: Academic Press; 2015. p. 425–36, CrossRef.

Singh V, Khurana A, Navik U, Allawadhi P, Bharani KK, Weiskirchen R. Apoptosis and pharmacological therapies for targeting thereof for cancer therapeutics. Sci. 2022; 4(2): 15, CrossRef.

Dechant MJ, Fellenberg J, Scheuerpflug CG, Ewerbeck V, Debatin KM. Mutation analysis of the apoptotic “death-receptors” and the adaptors TRADD and FADD/MORT-1 in osteosarcoma tumor samples and osteosarcoma cell lines. Int J Cancer. 2004; 109(5): 661–7, CrossRef.

Meiliana A, Dewi NM, Wijaya A. Cancer genetics and epigenetics in cancer risk assesment. Mol Cell Biomed Sci. 2021; 5(2): 41–61, CrossRef.


Copyright (c) 2022 The Prodia Education and Research Institute

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.


Indexed by:






The Prodia Education and Research Institute