Caffeic Acid Induces Apoptosis in MG-63 Osteosarcoma Cells via Protein Kinase C Delta (PKCδ) Translocation and Mitochondrial Membrane Potential Reduction

Ferry Sandra, Muhammad Ihsan Rizal, Caecilia Caroline Aliwarga, Jenifer Christy Hadimartana, Maria Celinna


BACKGROUND: Caffeic acid has been reported to activate caspases in MG-63 osteosarcoma cells, which can lead to apoptosis via both extrinsic and intrinsic apoptotic pathways. Translocation of protein kinase C delta (PKCδ), which reduces mitochondrial membrane potential (ΔΨm), is involved in apoptosis. The role of PKCδ translocation and ΔΨm alteration in caffeic acid-induced MG-63 cell apoptosis are largely unknown. Present study investigated the effect of caffeic acid on PKCδ translocation and ΔΨm in MG-63 cells.

METHODS: MG-63 cells were cultured and starved, followed by pretreatment with or without Z-VAD-FMK and treatment with or without 10 μg/mL caffeic acid. MG-63 cells were collected, lysed, and processed to obtain cytosolic and mitochondrial fractions. Each fraction was subjected to immunoblotting analysis by using anti-PKCδ antibody. Mitochondrial membrane potential (ΔΨm) was measured using flow cytometry.

RESULTS: Cytosolic PKCδ levels were higher than mitochondrial PKCδ levels in untreated and 1 h caffeic acid treatment groups. Inversely, cytosolic PKCδ levels were lower than the mitochondrial PKCδ levels after 6 and 12 h caffeic acid treatment. By Z-VAD-FMK pretreatment, cytosolic PKCδ levels were higher than mitochondrial PKCδ after 6 and 12 h caffeic acid treatment. After 6 h treatment with caffeic acid, ΔΨm was slightly shifted. More shifting occurred in MG-63 cells treated with caffeic acid for 12 h. The ΔΨm shifting was inhibited by Z-VAD-FMK pretreatment.

CONCLUSION: Caffeic acid could trigger apoptosis of MG-63 osteosarcoma cells by inducing PKCδ translocation to mitochondria and reducing ΔΨm, which might cause MMP.

KEYWORDS: caffeic acid, MG-63, osteosarcoma, PKCδ, mitochondrial membrane potential, mitochondrial membrane permeabilization, 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, Böhling TO, Cates JMM, Cleton-Jansen AM, Hogendoorn PCW, O’Donnell PG, et al. Osteosarcoma. In: Antonescu CR, Blay J, Bovee JVMG, Bridge JA, Cunha IW, Dei Tos AP, et al., editors. Soft Tissue and Bone Tumours. 5th ed. Lyon: International Agency for Research on Cancer; 2020. p. 403–9.

Bertin H, Gomez-Brouchet A, Rédini F. Osteosarcoma of the jaws: An overview of the pathophysiological mechanisms. Crit Rev Oncol Hematol. 2020; 156: 103126, CrossRef.

Lee RJ, Arshi A, Schwartz HC, Christensen RE. Characteristics and prognostic factors of osteosarcoma of the jaws: A retrospective cohort study. JAMA Otolaryngol Head Neck Surg. 2015; 141(5): 470–7, CrossRef.

Agrawal RR, Bhavthankar JD, Mandale MS, Patil PP. Osteosarcoma of jaw with varying histomorphologic patterns: Case report. J Orthop Case Rep. 2017; 7(1): 61–4, CrossRef.

Krishnamurthy A, Palaniappan R. Osteosarcomas of the head and neck region: A case series with a review of literature. J Maxillofac Oral Surg. 2018; 17(1): 38–43, CrossRef.

Rahman MN, Wijaya CR, Novalentina M. Survivin clinical features in cervical cancer. Mol Cell Biomed Sci. 2017; 1(1): 6–16, CrossRef.

Boon E, van der Graaf WTA, Gelderblom H, Tesselaar MET, van Es RJJ, Oosting SF, et al. Impact of chemotherapy on the outcome of osteosarcoma of the head and neck in adults: Osteosarcoma of the head and neck. Head Neck. 2017; 39(1): 140–6, CrossRef.

Chen Y, Gokavarapu S, Shen Q, Liu F, Cao W, Ling Y, et al. Chemotherapy in head and neck osteosarcoma: Adjuvant chemotherapy improves overall survival. Oral Oncol. 2017; 73: 124–31, CrossRef.

Sandra F. Targeting ameloblatoma into apoptosis. Indones Biomed J. 2018; 10(1): 35–9, CrossRef.

Mahanani ES, Arifin IN, Ihsan AN, Lukitasari Y, Sandra F. Parkia speciosa seeds ethanol extract as co-chemotherapeutic agent for doxorubicin toward tongue cancer. Indones Biomed J. 2022; 14(2): 186–92, 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.

Matboli M, Eissa S, Ibrahim D, Hegazy MGA, Imam SS, Habib EK. Caffeic acid attenuates diabetic kidney disease via modulation of autophagy in a high-fat diet/streptozotocin-induced diabetic rat. Sci Rep. 2017; 7(1): 2263, CrossRef.

Andrade S, Loureiro JA, Pereira MC. Caffeic acid for the prevention and treatment of Alzheimer’s disease: The effect of lipid membranes on the inhibition of aggregation and disruption of Aβ fibrils. Int J Biol Macromol. 2021; 190: 853–61, CrossRef.

Pelinson LP, Assmann CE, Palma TV, da Cruz IBM, Pillat MM, Mânica A, et al. Antiproliferative and apoptotic effects of caffeic acid on SK-Mel-28 human melanoma cancer cells. Mol Biol Rep. 2019; 46(2): 2085–92, CrossRef.

Teng YN, Wang CCN, Liao WC, Lan YH, Hung CC. Caffeic acid attenuates multi-drug resistance in cancer cells by inhibiting efflux function of human P-glycoprotein. Molecules. 2020; 25(2): 247, 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.

Sandra F, Rizal MI, Wahid AHA, Andajana M, Celinna M. Caffeic acid induces intrinsic apoptotic pathway in MG-63 osteosarcoma cells through Bid truncation and cytochrome c release. Indones Biomed J. 2022; 14(3): 323–8, CrossRef.

Baek JH, Yun HS, Kwon GT, Lee J, Kim JY, Jo Y, et al. PLOD3 suppression exerts an anti-tumor effect on human lung cancer cells by modulating the PKC-delta signaling pathway. Cell Death Dis. 2019; 10(3): 156, CrossRef.

Gurbuz N, Park MA, Dent P, Abdel Mageed AB, Sikka SC, Baykal A. Cystine dimethyl ester induces apoptosis through regulation of PKC-δ and PKC-ε in prostate cancer cells. Anticancer Agents Med Chem. 2015; 15(2): 217–27, CrossRef.

Brodie C, Blumberg PM. Regulation of cell apoptosis by protein kinase c δ. Apoptosis. 2003; 8(1): 19–27, CrossRef.

Kroemer G, Galluzzi L, Brenner C. Mitochondrial membrane permeabilization in cell death. Physiol Rev. 2007; 87(1): 99-163, 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.

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.

Chao MW, Chen TH, Huang HL, Chang YW, HuangFu WC, Lee YC, et al. Lanatoside C, a cardiac glycoside, acts through protein kinase Cδ to cause apoptosis of human hepatocellular carcinoma cells. Sci Rep. 2017; 7(1): 46134, CrossRef.

Rijo P, Simões MF, Francisco AP, Rojas R, Gilman RH, Vaisberg AJ, et al. Antimycobacterial metabolites from Plectranthus: Royleanone derivatives against Mycobacterium tuberculosis strains. Chem Biodivers. 2010; 7(4): 922–32, CrossRef.

Bessa C, Soares J, Raimundo L, Loureiro JB, Gomes C, Reis F, et al. Discovery of a small-molecule protein kinase Cδ-selective activator with promising application in colon cancer therapy. Cell Death Dis. 2018; 9(2): 23, CrossRef.

Mishra S, Vinayak M. Role of ellagic acid in regulation of apoptosis by modulating novel and atypical PKC in lymphoma bearing mice. BMC Complement Altern Med. 2015; 15: 281, CrossRef.

Wilkins LR, Brautigan DL, Wu H, Yarmohammadi H, Kubicka E, Serbulea V, et al. Cinnamic acid derivatives enhance the efficacy of transarterial embolization in a rat model of hepatocellular carcinoma. Cardiovasc Intervent Radiol. 2017; 40(3): 430–7, CrossRef.

Feriotto G, Tagliati F, Giriolo R, Casciano F, Tabolacci C, Beninati S, et al. Caffeic acid enhances the anti-leukemic effect of imatinib on chronic myeloid leukemia cells and triggers apoptosis in cells sensitive and resistant to imatinib. Int J Mol Sci. 2021; 22(4): 1644, CrossRef.

Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 1998; 17(6): 1675–87, CrossRef.

Hanahan D. Hallmarks of cancer: New dimensions. Cancer Discov. 2022; 12(1): 31–46, CrossRef.

Saragih CF, Rivany R, Sahil MF, Fadjrir F, Ardiansyah E, Yaznil MR, et al. The difference of Bax protein expression between endometrioma and ovarian carcinoma. Mol Cell Biomed Sci. 2019; 3(2): 95–9, CrossRef.

Chen LB. Mitochondrial membrane potential in living cells. Ann Rev Cell Biol. 1988; 4: 155–81, CrossRef.

Green DR, Evan GI. A matter of life and death. Cancer Cell. 2002; 1(1): 19–30, 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