Luteolin induces intrinsic apoptosis via inhibition of E6/E7 oncogenes and activation of extrinsic and intrinsic signaling pathways in HPV-18-associated cells

Abstract

Luteolin, a flavonoid extracted from a number of plants with recognized anticancer, anti-inflammatory and anti-oxidative activities, inhibits angiogenic processes and modulates multidrug resistance. However, the efficacy and mechanisms of action of this flavonoid agent are still undergoing study. In order to elucidate whether luteolin exhibits an anticancer effect in cervical cancer cells, HeLa cells were incubated with luteolin and apoptosis was assessed by observing nuclear morphological changes, and performing Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Cell cycle analysis, western blotting, RT-PCR and mitochondrial membrane potential measurements were also carried out. Luteolin showed a significant dose-dependent cytotoxic effect only in human papillomavirus (HPV)-positive cervical cancer cells, when compared to its effect on HPV-negative cervical cancer C33A cells. Expression levels of human papilloma virus E6 and E7 oncogenes were suppressed, those of related factors pRb and p53 were recovered and E2F5 was increased by luteolin treatment. Furthermore, luteolin enhanced the expression of death receptors and death receptor downstream factors such as Fas/FasL, DR5/TRAIL and FADD in HeLa cells, and activated caspase cascades. In particular, luteolin enhanced the activity of caspase-3 and -8 in a dose-dependent manner. Activation of caspase-3 induced caspase-8 activity and vice versa. Luteolin also induced mitochondrial membrane potential collapse and cytochrome c release, and inhibited Bcl-2 and Bcl-xL expression. In conclusion, luteolin exerts anticarcinogenic activity through inhibition of E6 and E7 expression and cross-activation of caspase-3 and -8. Taken together, these results suggest that luteolin induces inactivation of HPV-18 oncogene expression and apoptosis by activating the intrinsic and extrinsic pathways.

Characteristic Features of Cytotoxic Activity of Flavonoids on Human Cervical Cancer Cells

“…The major etiological factor in the formation of cervical cancer is the infection with human papillomavirus (HPV) and more than 99% of cervical tumors contain one or more of the oncogenic HPV genotypes (Lee et al, 2011;Di Domenico et al, 2012;Kim et al, 2012;Kim MS et al, 2013;Zhu et al, 2013;Garcia et al, 2014;Ham et al, 2014). There are more than 200 types of HPVs described to date from which about 15 are designed as high-risk strains according to the propensity of malignant progression of virus-associated lesions (Di Domenico et al, 2012;Yuan CH, 2012;Hirchaud et al, 2013;Moga et al, 2014).…”

“…Furthermore, jaceosidin is proved to specifically inhibit HPV16-harboring cervical carcinoma cell lines CaSki and SiHa and exert only very little or no inhibition in HPV18-positive HeLa cells and HPV-negative C33A cells (Lee et al, 2005 DOI:http://dx.doi.org/10.7314/APJCP.2014.15.19.8007 Characterisitics of Cytotoxicity of Flavonoids on Cervical Cancer Cells apoptosis of HPV18-positive HeLa cells exhibiting only mild cytotoxicity at high doses in HPV16-harboring cells and no effect on HPV-free C33A cells (Xie F et al, 2012;Krifa et al, 2013;Ham et al, 2014). It is probable that jaceosidin can specifically downregulate the E6 and E7 oncoproteins of HPV type 16 and luteolin those of HPV type 18.…”

Dealing Naturally with Stumbling Blocks on Highways and Byways of TRAIL Induced Signaling

“…Intrinsic pathway was activated via activation of caspase-3 by caspase-8. Moreover, efflux of cytochrome c from mitochondria was also noted alongwith inhibition of Bcl-2 and Bcl-xL (Ham et al, 2014). Luteolin treated 786-O renal cell carcinoma cells displayed a decrease in Mcl-1 and FLIP levels.…”

A Novel Mutant of Human Papillomavirus Type 18 E6E7 Fusion Gene and its Transforming Activity

“…E6 proteins induce the rapid degradation of p53 through ubiquitin-dependent proteolysis. The p53 tumor suppressor is not only required for normal cellular proliferation but also functions as a guardian of the human genome (Ham et al, 2014). E7 protein can bind and induce the degradation of the pRb protein and hence the pRb lost it ability as an antioncogene (Balsitis et al, 2006;Ham et al, 2014).…”

“…The p53 tumor suppressor is not only required for normal cellular proliferation but also functions as a guardian of the human genome (Ham et al, 2014). E7 protein can bind and induce the degradation of the pRb protein and hence the pRb lost it ability as an antioncogene (Balsitis et al, 2006;Ham et al, 2014). Since E6 and E7 are consistently expressed in most cervical cancers and their precursor lesions but absent in normal tissues, these viral oncoproteins represent effective targets for the development of specific therapeutic vaccines (Pang et al, 2013).…”