Abstract
Background: Hepatocellular carcinoma (HCC) is associated with a high mortality rate due to early recurrence and its metastasis features. To this day, effective treatment options for metastatic HCC remain a major challenge to patient treatment. Flavokawain B (FKB) is a naturally occurring chalcone molecule capable of providing effective therapy against this life-threatening disease.
Objective: This study investigated the anti-metastatic effects of FKB on the growth and development of metastatic HCC.
Methods: HepG2 cells were used in this study and a neutral red assay was performed to determine the IC50 value of FKB. Cell scratch and exclusion zone assays were performed to assess the rate of cell migration and invasion. Relative mRNA levels of UCK2, STAT3, VEGF and HIF-1α genes were quantified using RT-qPCR.
Results: FKB inhibited the proliferation of HepG2 cells at an IC50 value of 28 μM after 72 h of incubation. Its cytotoxic effect was confirmed to induce apoptosis through the phase-contrast inverted microscope. Cell migration and invasion were significantly inhibited at 7, 14, and 28 μM of FKB as compared to untreated cells. The inhibition in the cell migration significantly increased with the increasing concentrations of the bioactive compound. The relative expression levels of the UCK2 gene and its downstream genes, STAT3, VEGF and HIF-1α, were significantly downregulated after 72 h exposure to FKB treatment.
Conclusion: Our data suggest that FKB inhibited HepG2 proliferation and further suppressed its metastasis partly by regulating the STAT3/Hif-1α/VEGF signalling pathway. FKB could be a potential alternative and viable strategy against HCC.
Graphical Abstract
[1]
Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021; 71(3): 209-49.
[http://dx.doi.org/10.3322/caac.21660] [PMID: 33538338]
[http://dx.doi.org/10.3322/caac.21660] [PMID: 33538338]
[2]
Becker AK, Tso DK, Harris AC, Malfair D, Chang SD. Extrahepatic metastases of hepatocellular carcinoma: A spectrum of imaging findings. Can Assoc Radiol J 2014; 65(1): 60-6.
[http://dx.doi.org/10.1016/j.carj.2013.05.004] [PMID: 24239313]
[http://dx.doi.org/10.1016/j.carj.2013.05.004] [PMID: 24239313]
[3]
Yu YM, Cao YS, Wu Z, Huang R, Shen ZL. Colon metastasis from hepatocellular carcinoma: a case report and literature review. World J Surg Oncol 2020; 18(1): 189.
[http://dx.doi.org/10.1186/s12957-020-01960-2] [PMID: 32723336]
[http://dx.doi.org/10.1186/s12957-020-01960-2] [PMID: 32723336]
[4]
BalajiSubramanian S, Sathiya K, Balaji K, Thirunavukarasu M, Phanikiran S, Rela M. Re-irradiation after stereotactic body radiotherapy for spine metastases from hepatocellular carcinoma: a case report. Rep Pract Oncol Radiother 2021; 26(6): 1060-5.
[http://dx.doi.org/10.5603/RPOR.a2021.0098] [PMID: 34992882]
[http://dx.doi.org/10.5603/RPOR.a2021.0098] [PMID: 34992882]
[5]
Tanoue K, Kawasaki Y, Yamasaki Y, et al. Postoperative recurrence with right cervical lymph node metastasis in hepatocellular carcinoma: a case report. Surg Case Rep 2021; 7(1): 260.
[http://dx.doi.org/10.1186/s40792-021-01352-y] [PMID: 34918186]
[http://dx.doi.org/10.1186/s40792-021-01352-y] [PMID: 34918186]
[6]
Obana A, Sato Y, Koyama M, et al. [A Case of Multiple Metachronous Metastases from Hepatocellular Carcinoma to Thoracic Spine and Right Adrenal Invading Right Kidney with Tumor Thrombus in the Inferior Vena Cava That Was Treated with Chemotherapy, Radiotherapy, Intravascular Therapy, and Surgery]. Gan To Kagaku Ryoho 2021; 48(13): 2088-90.
[PMID: 35045502]
[PMID: 35045502]
[7]
Wen T, Jin C, Facciorusso A, et al. Multidisciplinary management of recurrent and metastatic hepatocellular carcinoma after resection: an international expert consensus. Hepatobiliary Surg Nutr 2018; 7(5): 353-71.
[http://dx.doi.org/10.21037/hbsn.2018.08.01] [PMID: 30498711]
[http://dx.doi.org/10.21037/hbsn.2018.08.01] [PMID: 30498711]
[8]
Cha C, Fong Y, Jarnagin WR, Blumgart LH, DeMatteo RP. Predictors and patterns of recurrence after resection of hepatocellular carcinoma. J Am Coll Surg 2003; 197(5): 753-8.
[http://dx.doi.org/10.1016/j.jamcollsurg.2003.07.003] [PMID: 14585409]
[http://dx.doi.org/10.1016/j.jamcollsurg.2003.07.003] [PMID: 14585409]
[9]
Zhang H, Zhang W, Jiang L, Chen Y. Recent advances in systemic therapy for hepatocellular carcinoma. Biomark Res 2022; 10(1): 3.
[http://dx.doi.org/10.1186/s40364-021-00350-4] [PMID: 35000616]
[http://dx.doi.org/10.1186/s40364-021-00350-4] [PMID: 35000616]
[10]
Zhou Q, Jiang H, Zhang J, et al. Uridine-cytidine kinase 2 promotes metastasis of hepatocellular carcinoma cells via the Stat3 pathway. Cancer Manag Res 2018; 10: 6339-55.
[http://dx.doi.org/10.2147/CMAR.S182859] [PMID: 30568496]
[http://dx.doi.org/10.2147/CMAR.S182859] [PMID: 30568496]
[11]
Cai J, Sun X, Guo H, et al. Non-metabolic role of UCK2 links EGFR-AKT pathway activation to metastasis enhancement in hepatocellular carcinoma. Oncogenesis 2020; 9(12): 103.
[http://dx.doi.org/10.1038/s41389-020-00287-7] [PMID: 33277463]
[http://dx.doi.org/10.1038/s41389-020-00287-7] [PMID: 33277463]
[12]
Malami I, Abdul AB. Involvement of the uridine cytidine kinase 2 enzyme in cancer cell death: A molecular crosstalk between the enzyme and cellular apoptosis induction. Biomed Pharmacother 2019; 109: 1506-10.
[http://dx.doi.org/10.1016/j.biopha.2018.10.200] [PMID: 30551402]
[http://dx.doi.org/10.1016/j.biopha.2018.10.200] [PMID: 30551402]
[13]
Malami I, Abdul A, Abdullah R, Bt Kassim N, Waziri P, Christopher Etti I. in silico Discovery of Potential Uridine-Cytidine Kinase 2 Inhibitors from the Rhizome of Alpinia mutica. Molecules 2016; 21(4): 417.
[http://dx.doi.org/10.3390/molecules21040417] [PMID: 27070566]
[http://dx.doi.org/10.3390/molecules21040417] [PMID: 27070566]
[14]
Hseu YC, Chiang YC, Vudhya Gowrisankar Y, et al. The in vitro and in vivo anticancer properties of chalcone flavokawain B through induction of rosmediated apoptotic and autophagic cell death in human melanoma cells. Cancers (Basel) 2020; 12(10): 2936.
[http://dx.doi.org/10.3390/cancers12102936] [PMID: 33053749]
[http://dx.doi.org/10.3390/cancers12102936] [PMID: 33053749]
[15]
Malami I, Muhammad A, Etti IC, Waziri PM, Alhassan AM. An in silico approach in predicting the possible mechanism involving restoration of wild-type p53 functions by small molecular weight compounds in tumor cells expressing R273H mutant p53. EXCLI J 2017; 16: 1276-87.
[http://dx.doi.org/10.17179/excli2017-299] [PMID: 29333130]
[http://dx.doi.org/10.17179/excli2017-299] [PMID: 29333130]
[16]
Malami I, Muhammad A, Abubakar IB, Alhassan AM. Expression of Wild-Type p53 by Curcumin, Alpinetin and Flavokawain B in Colorectal Cancer cells Expressing R273H Mutant p53. Nigerian Journal of Basic and Applied Sciences 2020; 27(2): 88-94.
[http://dx.doi.org/10.4314/njbas.v27i2.12]
[http://dx.doi.org/10.4314/njbas.v27i2.12]
[17]
Alitheen NB, Abu N, Mohamad NE, et al. in vivo antitumor and antimetastatic effects of flavokawain B in 4T1 breast cancer cell-challenged mice. Drug Des Devel Ther 2015; 9: 1401-17.
[http://dx.doi.org/10.2147/DDDT.S67976] [PMID: 25834398]
[http://dx.doi.org/10.2147/DDDT.S67976] [PMID: 25834398]
[18]
Repetto G, del Peso A, Zurita JL. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 2008; 3(7): 1125-31.
[http://dx.doi.org/10.1038/nprot.2008.75] [PMID: 18600217]
[http://dx.doi.org/10.1038/nprot.2008.75] [PMID: 18600217]
[19]
Liang CC, Park AY, Guan JL. in vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2007; 2(2): 329-33.
[http://dx.doi.org/10.1038/nprot.2007.30] [PMID: 17406593]
[http://dx.doi.org/10.1038/nprot.2007.30] [PMID: 17406593]
[20]
Hua R, Pei Y, Gu H, Sun Y, He Y. Antitumor effects of flavokawain-B flavonoid in gemcitabine-resistant lung cancer cells are mediated via mitochondrial-mediated apoptosis, ROS production, cell migration and cell invasion inhibition and blocking of PI3K/AKT Signaling pathway. J BUON 2020; 25(1): 262-7.
[PMID: 32277640]
[PMID: 32277640]
[21]
Hseu YC, Huang YC, Thiyagarajan V, et al. Anticancer activities of chalcone flavokawain B from Alpinia pricei Hayata in human lung adenocarcinoma (A549) cells via induction of reactive oxygen species-mediated apoptotic and autophagic cell death. J Cell Physiol 2019; 234(10): 17514-26.
[http://dx.doi.org/10.1002/jcp.28375] [PMID: 30847898]
[http://dx.doi.org/10.1002/jcp.28375] [PMID: 30847898]
[22]
Wang J, Qi Q, Zhou W, et al. Inhibition of glioma growth by flavokawain B is mediated through endoplasmic reticulum stress induced autophagy. Autophagy 2018; 14(11): 2007-22.
[http://dx.doi.org/10.1080/15548627.2018.1501133] [PMID: 30025493]
[http://dx.doi.org/10.1080/15548627.2018.1501133] [PMID: 30025493]
[23]
Malami I, Abdul AB, Abdullah R, et al. Crude extracts, flavokawain B and alpinetin compounds from the rhizome of Alpinia mutica induce cell death viaUCK2 enzyme inhibition and in turn reduce 18S rRNA biosynthesis in HT-29 cells. PLoS One 2017; 12(1): e0170233.
[http://dx.doi.org/10.1371/journal.pone.0170233] [PMID: 28103302]
[http://dx.doi.org/10.1371/journal.pone.0170233] [PMID: 28103302]
[24]
Hseu YC, Lin RW, Shen YC, et al. Flavokawain b and doxorubicin work synergistically to impede the propagation of gastric cancer cells via ros-mediated apoptosis and autophagy pathways. Cancers (Basel) 2020; 12(9): 2475.
[http://dx.doi.org/10.3390/cancers12092475] [PMID: 32882870]
[http://dx.doi.org/10.3390/cancers12092475] [PMID: 32882870]
[25]
Lee JJ, Koh KN, Park CJ, Jang S, Im HJ, Kim N. The combination of flavokawain B and daunorubicin induces apoptosis in human myeloid leukemic cells by modifying NF-ĸB. Anticancer Res 2018; 38(5): 2771-8.
[http://dx.doi.org/10.21873/anticanres.12520] [PMID: 29715098]
[http://dx.doi.org/10.21873/anticanres.12520] [PMID: 29715098]
[26]
Waziri PM, Abdullah R, Yeap SK, et al. Clausenidin from Clausena excavata induces apoptosis in hepG2 cells via the mitochondrial pathway. J Ethnopharmacol 2016; 194: 549-58.
[http://dx.doi.org/10.1016/j.jep.2016.10.030] [PMID: 27729282]
[http://dx.doi.org/10.1016/j.jep.2016.10.030] [PMID: 27729282]
[27]
Waziri PM, Abdullah R, Yeap SK, et al. Clausenidin induces caspase-dependent apoptosis in colon cancer. BMC Complement Altern Med 2016; 16(1): 256.
[http://dx.doi.org/10.1186/s12906-016-1247-1] [PMID: 27473055]
[http://dx.doi.org/10.1186/s12906-016-1247-1] [PMID: 27473055]
[28]
Etti I, Rasedee A, Mohd Hashim N, et al. Artonin E induces p53-independent G1 cell cycle arrest and apoptosis through ROS-mediated mitochondrial pathway and livin suppression in MCF-7 cells. Drug Des Devel Ther 2017; 11: 865-79.
[http://dx.doi.org/10.2147/DDDT.S124324] [PMID: 28356713]
[http://dx.doi.org/10.2147/DDDT.S124324] [PMID: 28356713]
[29]
Etti IC, Abdullah R, Kadir A, et al. The molecular mechanism of the anticancer effect of Artonin E in MDA-MB 231 triple negative breast cancer cells. PLoS One 2017; 12(8): e0182357.
[http://dx.doi.org/10.1371/journal.pone.0182357] [PMID: 28771532]
[http://dx.doi.org/10.1371/journal.pone.0182357] [PMID: 28771532]
[30]
Wang HQ, Man QW, Huo FY, et al. STAT3 pathway in cancers: Past, present, and future. MedComm 2022; 3(2): e124.
[http://dx.doi.org/10.1002/mco2.124] [PMID: 35356799]
[http://dx.doi.org/10.1002/mco2.124] [PMID: 35356799]
[31]
Malami I, Muhammad A, Abubakar IB, et al. 5,6-dehydrokawain from the rhizome of Alpinia mutica Roxb. induced proangiogenic tumour-derived VEGF of HT-29 colorectal cancer. Nat Prod Res 2018; 32(24): 2964-7.
[http://dx.doi.org/10.1080/14786419.2017.1392954] [PMID: 29052437]
[http://dx.doi.org/10.1080/14786419.2017.1392954] [PMID: 29052437]
[32]
Niu G, Wright KL, Huang M, et al. Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis. Oncogene 2002; 21(13): 2000-8.
[http://dx.doi.org/10.1038/sj.onc.1205260] [PMID: 11960372]
[http://dx.doi.org/10.1038/sj.onc.1205260] [PMID: 11960372]
[33]
Zhang PC, Liu X, Li MM, et al. AT-533, a novel Hsp90 inhibitor, inhibits breast cancer growth and HIF-1α/VEGF/VEGFR-2-mediated angiogenesis in vitro and in vivo . Biochem Pharmacol 2020; 172: 113771.
[http://dx.doi.org/10.1016/j.bcp.2019.113771] [PMID: 31863779]
[http://dx.doi.org/10.1016/j.bcp.2019.113771] [PMID: 31863779]
