β-Hydroxyisovalerylshikonin promotes reactive oxygen species production in HCT116 colon cancer cells, leading to caspase-mediated apoptosis

Dilshara, Matharage Gayani,Karunarathne, Wisurumuni Arachchilage Hasitha Maduranga,Molagoda, Ilandarage Menu Neelaka,Kang, Chang-Hee,Jeong, Jin-Woo,Choi, Yung Hyun,Kim, Gi-Young Elsevier 2018 Revista brasileira de farmacognosia Vol.28 No.3

<P><B>Abstract</B></P> <P>Although β-hydroxyisovalerylshikonin is suggested as a potential therapeutic agent for preventing various cancers, the underlying molecular mechanisms are not completely understood. In the present study, we investigated whether β-hydroxyisovalerylshikonin enhances apoptosis by triggering reactive oxygen species production in colon cancer HCT116 cells. β-Hydroxyisovalerylshikonin significantly inhibited the viability of HCT116 cells with maximum inhibition at 4μM. Furthermore, treatment with β-hydroxyisovalerylshikonin subsequently increased sub-G<SUB>1</SUB> cells and annexin-V<SUP>+</SUP> cell population. Additionally, pretreatment with the caspase-8 inhibitor, z-IETD-fmk, and the caspase-9 inhibitor, z-LETD-fmk, significantly decreased β-hydroxyisovalerylshikonin-induced apoptosis, suggesting that β-hydroxyisovalerylshikonin promotes apoptosis through both the intrinsic and the extrinsic apoptotic pathways by activating caspase-8 and caspase-9. We also found that mitochondria played an important role in β-hydroxyisovalerylshikonin-mediated apoptosis via the intrinsic pathway. Accordingly, β-hydroxyisovalerylshikonin-induced reactive oxygen species production was evident after treatment with β-hydroxyisovalerylshikonin, and pretreatment with reactive oxygen species inhibitors, <I>N</I>-acetyl-<SMALL>L</SMALL>-cysteine and glutathione, significantly decreased β-hydroxyisovalerylshikonin-induced reactive oxygen species production, resulting in inhibition of apoptosis, which suggests that ROS generation is required for β-hydroxyisovalerylshikonin-mediated apoptosis. Taken together, these results demonstrated that the apoptotic effect of β-hydroxyisovalerylshikonin is enhanced in colon cancer HCT116 cells via reactive oxygen species generation and triggering of the caspase pathways, indicating that β-hydroxyisovalerylshikonin has potential as a therapeutic in the treatment of colon cancers.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Methanol extract of Codium fragile inhibits tumor necrosis factor-α-induced matrix metalloproteinase-9 and invasiveness of MDA-MB-231 cells by suppressing nuclear factor-κB activation

Dilshara, M.G.,Jayasooriya, R.G.P.T.,Kang, C.H.,Choi, Y.H.,Kim, G.Y. Elsevier 2016 Asian Pacific journal of tropical medicine Vol.9 No.6

<P>Objective: To evaluate whether the methanol extract of Codium fragile (MECF) regulates tumor necrosis factor-alpha (TNF-alpha)-induced invasion of human breast cancer MDA-MB-231 cells by suppressing matrix metalloproteinase-9 (MMP-9). Methods: Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were performed to analyze the expression of MMP-9 and nuclear factor-kappa B (NF-kappa B) subunits, p65 and p50, and I kappa B in MDA-MB-231 cells. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used for cell viability. MMP-9 activity and invasion were measured by gelatin zymography and a matrigel invasion assay, respectively. NF-kappa B activity was measured by an electrophoretic mobility shift assay and luciferase activity. Results: MECF had no effect on cell viability up to a concentration of 100 mg/mL in human breast cancer MDA-MB-231 cells regardless of the presence of TNF-alpha. MDA-MB-231 cells that were stimulated with TNF-alpha showed a marked increase of invasion compared to the untreated control, whereas pretreatment with MECF downregulated the TNF-alpha-induced invasion of MDA-MB-231 cells. Additionally, zymography, western blot analysis, and RT-PCR confirmed that MECF decreased TNF-alpha-induced MMP-9 expression and activity which is a key regulator for cancer invasion. According to an electrophoretic morbidity shift assay, pretreatment with MECF in MDA-MB-231 cells significantly decreased the TNF-alpha-induced DNA-binding activity of NF-kappa B, which is an important transcription factor for regulating cancer invasion-related genes such as MMP-9. Furthermore, treatment with MECF sustained the expression of p65 and p50 in response to TNF-alpha in the cytosolic compartment. The luciferase assay demonstrated that MECF attenuated TNF-alpha-induced NF-kappa B luciferase activity. Conclusion: MECF exhibited its anti-invasive capability by downregulating TNF-alpha-induced MMP-9 expression, resulting from the suppression of NF-kappa B activity in the human breast cancer cell line MDA-MB-231.</P>

Glutamine deprivation sensitizes human breast cancer MDA-MB-231 cells to TRIAL-mediated apoptosis

Dilshara, Matharage Gayani,Jeong, Jin-Woo,Prasad Tharanga Jayasooriya, Rajapaksha Gedara,Neelaka Molagoda, Ilandarage Menu,Lee, Seungheon,Park, Sang Rul,Choi, Yung Hyun,Kim, Gi-Young Elsevier 2017 Biochemical and biophysical research communication Vol. No.

<P><B>Abstract</B></P> <P>Tumor cell metabolism is a promising target for various cancer treatments. Apart from aerobic glycolysis, cancer cell growth is dependent on glutamine (Gln) supply, leading to their survival and differentiation. Therefore, we examined whether treatment with TNF-related apoptosis-inducing ligand (TRAIL) sensitizes MDA-MB-231 cells to apoptosis under Gln deprivation condition (TRAIL/Gln deprivation). Gln deprivation decreased cell proliferation as expected, but did not induce remarkable cell death. TRAIL/Gln deprivation, however, significantly increased growth inhibition and morphological shrinkage of MDA-MB-231 cells compared to those induced by treatment with either Gln deprivation or TRAIL alone. Moreover, TRAIL/Gln deprivation upregulated the apoptotic sub-G<SUB>1</SUB> phase accompanied with a remarkable decrease of pro-caspase-3, pro-caspase-9, and anti-apoptotic xIAP, and Bcl-2. Increased cleavage of PARP and pro-apoptotic Bid protein expression suggests that TRAIL/Gln deprivation triggers mitochondrion-mediated apoptosis in MDA-MB-231 cells. Additionally, TRAIL/Gln deprivation upregulated the expression of endoplasmic reticulum (ER) stress markers such as ATF4 and phosphorylated eIF2α, thereby enhancing the C/EBP homologous protein (CHOP) protein level. Transient knockdown of <I>CHOP</I> partically reversed TRAIL/Gln deprivation-mediated apoptosis. Accordingly, TRAIL/Gln deprivation enhanced the expression of death receptor 5 (DR5) and transient knockdown of <I>DR5</I> completely restored TRAIL/Gln deprivation-mediated apoptosis. Taken together, our results suggest that Gln deprivation conditions can be used for the development of new therapies for TRAIL-resistant cancers.</P>

Mangiferin inhibits tumor necrosis factor-α-induced matrix metalloproteinase-9 expression and cellular invasion by suppressing nuclear factor-κB activity

( Matharage Gayani Dilshara ),( Chang Hee Kang ),( Yung Hyun Choi ),( Gi Young Kim ) 생화학분자생물학회(구 한국생화학분자생물학회) 2015 BMB Reports Vol.48 No.10

We investigated the effects of mangiferin on the expression and activity of metalloproteinase (MMP)-9 and the invasion of tumor necrosis factor (TNF)-α-stimulated human LNCaP prostate carcinoma cells. Reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis showed that mangiferin significantly reversed TNF-α-induced mRNA and protein expression of MMP-9 expression. Zymography data confirmed that stimulation of cells with TNF-α significantly increased MMP-9 activity. However, mangiferin substantially reduced the TNF-α-induced activity of MMP-9. Additionally, a matrigel invasion assay showed that mangiferin significantly reduced TNF-α-induced invasion of LNCaP cells. Compared to untreated controls, TNF-α-stimulated LNCaP cells showed a significant increase in nuclear factor-κB (NF-κB) luciferase activity. However, mangiferin treatment markedly decreased TNF-α-induced NF-κB luciferase activity. Furthermore, mangiferin suppressed nuclear translocation of the NF-κB subunits p65 and p50. Collectively, our results indicate that mangiferin is a potential anti-invasive agent that acts by suppressing NF-κB-mediated MMP-9 expression. [BMB Reports 2015; 48(10): 559-564]

Camptothecin enhances c-Myc-mediated endoplasmic reticulum stress and leads to autophagy by activating Ca²⁺-mediated AMPK

Jayasooriya, Rajapaksha Gedara Prasad Tharanga,Dilshara, Matharage Gayani,Karunarathne, Wisurumuni Arachchilage Hasitha Maduranga,Molagoda, Ilandarage Menu Neelaka,Choi, Yung Hyun,Kim, Gi-Young Elsevier 2018 Food and chemical toxicology Vol.121 No.-

<P><B>Abstract</B></P> <P>Camptothecin (CPT) from <I>Camptotheca acuminate</I> was discovered for anticancer drugs, which targets topoisomease I. However, whether CPT regulates c-Myc expression has not been understood in endoplasmic reticulum (ER) stress and autophagy. In this study, we found that CPT enhanced c-Myc expression and that the transient knockdown of <I>c-Myc</I> abrogated reactive oxygen species (ROS) generation, which resulted in the accumulation of ER stress-regulating proteins, such as PERK, eIF2α, ATF4, and CHOP. Moreover, the transfection of <I>eIF2α</I>-targeted siRNA attenuated CPT-induced autophagy and decreased the levels of Beclin-1 and Atg7, which indicated that CPT upregulated ER stress-mediated autophagy. In addition, CPT phosphorylated AMPK in response to intracellular Ca<SUP>2+</SUP> release. Ca<SUP>2+</SUP> chelators, ethylene glycol tetraacetic acid and a CaMKII inhibitor, K252a, decreased CPT-induced Beclin-1 and Atg7, and downregulated AMPK phosphorylation, which suggested that CPT-induced Ca<SUP>2+</SUP> release leads to the activation of autophagy through CaMKII-mediated AMPK phosphorylation. CPT also phosphorylated JNK and activated the DNA-binding activity of AP-1; furthermore, knockdown of <I>JNK</I> abolished the expression level of Beclin-1 and Atg7, which implied that the JNK-AP-1 pathway was a potent mediator of CPT-induced autophagy. Our findings indicated that CPT promoted c-Myc-mediated ER stress and ROS generation, which enhances autophagy via the Ca<SUP>2+</SUP>-AMPK and JNK-AP-1 pathways.</P> <P><B>Highlights</B></P> <P> <UL> <LI> CPT induces c-Myc-mediated ROS formation, leading to CHOP expression. </LI> <LI> c-Myc positively regulates CPT-induced ER stress by increasing ROS generation. </LI> <LI> CPT promotes autophagy formation as a result of ER stress. </LI> <LI> CPT promotes autophagy through increased intracellular Ca2+ release. </LI> <LI> CPT induces JNK-mediated autophagy by enhancing AP-1 activity. </LI> </UL> </P>

Fulvic acid promotes extracellular anti-cancer mediators from RAW 264.7 cells, causing to cancer cell death <i>in vitro</i>

Jayasooriya, Rajapaksha Gedara Prasad Tharanga,Dilshara, Matharage Gayani,Kang, Chang-Hee,Lee, Seungheon,Choi, Yung Hyun,Jeong, Yong Kee,Kim, Gi-Young Elsevier 2016 INTERNATIONAL IMMUNOPHARMACOLOGY Vol.36 No.-

<P><B>Abstract</B></P> <P>Fulvic acid (FA) is known to promote electrochemical balance as a donor or a receptor possessing many biomedical functions. Nevertheless, the effect of FA on the anti-cancer activity has not been elucidated. In the current study, we first isolated FA from humus and investigated whether FA regulates immune-stimulating functions, such as production of nitric oxide (NO), in RAW 264.7 cells. Our data showed that FA slightly enhances cell viability in a dose-dependent manner and secretion of NO from RAW 264.7 cells. It upregulated the protein and mRNA expression of inducible NO synthesis (iNOS). In addition, FA enhanced the DNA-binding activity of nuclear factor-κB (NF-κB) in RAW 264.7 cells; the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) effectively attenuated the expression of FA-stimulated iNOS, suggesting that FA stimulates NF-κB to promote iNOS and NO production. Finally, FA-stimulated culture media (FA-CM) from RAW 264.7 cells were collected and MCA-102 fibrosarcoma cells were cultured in this media. The FA-CM augmented MCA-102 fibrosarcoma cell apoptosis; however, an NO inhibitor <I>N</I> <SUP>G</SUP>-monomethyl-<SMALL>L</SMALL>-arginine (NMMA) slightly inhibited the FA-CM-mediated MCA-102 fibrosarcoma cell apoptosis, which was accompanied by low levels of NO. In the present study, we found that FA induces the generation of NO and iNOS in RAW 264.7 cells by inducing NF-κB activation; however, NO did not significantly stimulate MCA-102 fibrosarcoma cell apoptosis in the current study. In addition, FA-CM enhanced cell death in various human cancer cells such as Hep3B, LNCaP, and HL60. Taken together, FA most likely stimulates immune-modulating molecules such as NO and induces cancer cell apoptosis.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Fulvic acid is isolated from humus by acid-base extraction methods. </LI> <LI> Fulvic acid increases proliferation of RAW 264.7 macrophage cells. </LI> <LI> Fulvic acid upregulates the expression of <I>iNOS</I> and NO by inducing NF-κB activity. </LI> <LI> Fulvic acid-stimulated medium of RAW 264.7 macrophages increases apoptosis of MCA-102 fibrosarcoma cells. </LI> </UL> </P>

Anti-inflammatory and anti-cancer activities of sterol rich fraction of cultured marine microalga Nannochloropsis oculata

Sanjeewa, Kalu Kapuge Asanka,Fernando, Ilekuttige Priyan Shanura,Samarakoon, Kalpa W.,Lakmal, Hetti Handi Chaminda,Kim, Eun-A,Kwon, O-Nam,Dilshara, Matharage Gayani,Lee, Joon-Baek,Jeon, You-Jin The Korean Society of Phycology 2016 ALGAE Vol.31 No.3

Five fractions separated from Nannochloropsis oculata using solvent-solvent partition chromatography of 80% methanolic extract of N. oculata (NOM) followed by the open silica column chromatography of its hexane fraction (NOMH) for the anti-inflammatory on RAW 264.7 cells and anti-cancer activities on HL-60, A-549, HEP-3B, HCT-116, and SW-480 cancer cells. All the five fractions showed potential anti-inflammatory activities against lipopolysaccharide-stimulated RAW 264.7 macrophages cells with IC₅₀ values less than 6.25 μg mL⁻¹. Moreover, 90% n-hexane column elution of NOMH (NOMH90) down-regulated lipopolysaccharide-stimulated protein levels of inducible nitric oxide synthase and cyclooxygenase-2. Furthermore, NOMH90 showed marked cytotoxic effect on the HL-60 cells with IC₅₀ value of 23.58 ± 0.09 μg mL⁻¹. In addition, Hoechst 33342 cell permeable dye used to visualize the apoptosis nucleus and cell cycle analysis measured Sub-G1 DNA contents to confirm reduction of the cell viability in NOMH90 treated cells due to induction of apoptosis in HL60. These results are quite related to the phytosterol contents of the NOMH fractions and the results suggest N. oculata extracts might be useful as potential sources of natural anti-inflammatory and anti-cancer compounds. In conclusion, the sterol content in N. oculata might provide a promising role in future medicines in anti-inflammatory and anti-cancer.