TNF-α Regulates Potassium Cyanate-induced Apoptosis via NF-κB Activation in HCT 116 Cells

Eun Ju Yang,Jeong Hyun Chang 대한의생명과학회 2014 Biomedical Science Letters Vol.20 No.1

Potassium cyanate (KOCN) that is known as an inducer of the protein carbamylation is an inorganic compound and is the conjugate based of cyanic acid (HOCN). Based on these studies, we confirmed that KOCN induces the apoptosis of the human colorectal cancer cell line, HCT 116 cells, by various mitochondrial pathways. To investigate other mechanisms of KOCN-mediated apoptosis, in the present study, we examined KOCN-induced cytokines production in HCT 116 cells and identified the intracellular signaling pathway in these processes. We first demonstrated that KOCN considerably increased the cell apoptosis via intracellular Ca<SUP>2+</SUP> signaling, mitochondrial dysfunction and ROS production. And then we examined TNF-α and IL-1β levels mediated by KOCN in HCT 116 cells. Although IL-1β was not involved in KOCN-mediated HCT 116 cell apoptosis, the release of TNF-α was mediated by KOCN in HCT 116 cells via NF-κB activation. Apoptosis was also enhanced by incubation with supernatants from HCT 116 cells after KOCN treatment and this effect was partially reduced by BAY 11-7085 pre-treated supernatant. Taken together, our results indicate that KOCN-induced apoptosis in HCT 116 cells is dependent on the releases of TNF-α and the increased factors and that the mechanism involves the activation of NF-κB.

The Extract of Pseudomonas aeruginosa Induces the Apoptosis of the Human Colorectal Cancer Cell Line, HCT 116 Cells, via Mitochondrial Pathway

Eun Ju Yang,Jeong Hyun Chang 대한의생명과학회 2012 Biomedical Science Letters Vol.18 No.1

Although there are many potential cytotoxic molecules released from bacteria, the role of these molecules on the apoptosis of various cancer cells is not well understood. Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative, aerobic and rod-shaped bacterium, and has a number of virulence factors. To understand the cytotoxic effect of bacterial extracts on the colorectal cancer cell line, HCT 116 cells, we examined alteration of the cell viability, proliferation, cell cycle and apoptosis of HCT 116 cells after treatment with extract of P. aeruginosa (PaE). These cytotoxicity of PaE occurred in a time- and a dose-dependent manners. In addition, PaE arrested the cell cycle of HCT 116 cell in a timedependent manner. PaE inhibited the protein levels of Bcl-2 and induced the release of cytochrome c from mitochondria of HCT 116 cells. The decrease of procaspase-3 was induced by the treatment of PaE. These results indicate that PaE has a cytotoxicity in HCT 116 cells via the induction of apoptosis associated with mitochondrial pathway. Therefore, PaE may used as the potential target for the treatment of colorectal cancer.

Characterization of sphere-forming HCT116 clones by whole RNA sequencing

Eunkyung Chung,Inkyung Oh,Kil Yeon Lee 대한외과학회 2016 Annals of Surgical Treatment and Research(ASRT) Vol.90 No.4

Purpose: To determine CD133<SUP>+</SUP> cells defined as cancer stem cells (CSCs) in colon cancer, we examined whether CD133<SUP>+</SUP> clones in HCT116 demonstrate known features of CSCs like sphere-forming ability, chemodrug-resistance, and metastatic potential. Methods: Magnetic cell isolation and cell separation demonstrated that <1% of HCT116 cells expressed CD133, with the remaining cells being CD133<SUP>-</SUP> clones. In colon cancer cells, radioresistance is also considered a CSC characteristic. We performed clonogenic assay using 0–4 Gy g-irradiation. Results: Interestingly, there were no differences between HCT116 parental and HCT116 CD133<SUP>+</SUP> clones when the cells comprised 0.5% of the total cells, and CD133<SUP>-</SUP> clone demonstrated radiosensitive changes compared with parental and CD133<SUP>+</SUP> clones. Comparing gene expression profiles between sphere-forming and nonforming culture conditions of HCT116 subclones by whole RNA sequencing failed to obtain specific genes expressed in CD133<SUP>+</SUP> clones. Conclusion: Despite no differences of gene expression profiles in monolayer attached culture conditions of each clone, sphere-forming conditions of whole HCT116 subclones, parental, CD133<SUP>+</SUP>, and CD133<SUP>-</SUP> increased 1,761 coding genes and downregulated 1,384 genes related to CSCs self-renewal and survival. Thus, spheroid cultures of HCT116 cells could be useful to expand colorectal CSCs rather than clonal expansion depending on CD133 expressions.

Ginsenoside Rh2 inhibiting HCT116 colon cancer cell proliferation through blocking PDZ-binding kinase/T-LAK cell-originated protein kinase

Yang, Jianjun,Yuan, Donghong,Xing, Tongchao,Su, Hongli,Zhang, Shengjun,Wen, Jiansheng,Bai, Qiqiang,Dang, Dongmei The Korean Society of Ginseng 2016 Journal of Ginseng Research Vol.40 No.4

Background: Ginsenoside Rh2 (GRh2) is the main bioactive component in American ginseng, a commonly used herb, and its antitumor activity had been studied in previous studies. PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK), a serine/threonine protein kinase, is highly expressed in HCT116 colorectal cancer cells. Methods: We examined the effect of GRh2 on HCT116 cells ex vivo. Next, we performed in vitro binding assay and in vitro kinase assay to search for the target of GRh2. Furthermore, we elucidated the underlying molecular mechanisms for the antitumor effect of GRh2 ex vivo and in vivo. Results: The results of our in vitro studies indicated that GRh2 can directly bind with PBK/TOPK and GRh2 also can directly inhibit PBK/TOPK activity. Ex vivo studies showed that GRh2 significantly induced cell death in HCT116 colorectal cancer cells. Further mechanistic study demonstrated that these compounds inhibited the phosphorylation levels of the extracellular regulated protein kinases 1/2 (ERK1/2) and (H3) in HCT116 colorectal cancer cells. In vivo studies showed GRh2 inhibited the growth of xenograft tumors of HCT116 cells and inhibited the phosphorylation levels of the extracellular regulated protein kinases 1/2 and histone H3. Conclusion: The results indicate that GRh2 exerts promising antitumor effect that is specific to human HCT116 colorectal cancer cells through inhibiting the activity of PBK/TOPK.

백두옹탕(白頭翁湯)의 대장암 세포주 HCT-116 항암효과와 세포자멸사에 관한 연구

김종욱,문구,박찬희,이정한,지혜민,Kim, Jong-Uk,Moon, Goo,Park, Chan-Ny,Lee, Jeong-Han,Ji, Hye-Min 대한한방내과학회 2010 大韓韓方內科學會誌 Vol.31 No.2

Objectives : To investigate the anti-cancer effect of Baekduong-tang(BDOT) against cancer cells, the signaling pathway of apoptosis was explored in human colon cancer cells. Materials and Methods : Human colon cancer cell lines, including HT-29 and HCT-116 cells, were used. Cell viability was measured by MTT assay. Apoptosis was determined by DAPI nuclei staining and flow cytometry in HCT-116 cells treated with 0.25 mg/$m{\ell}$ Baekduong-tang for 48 hrs. Results : Baekduong-tang induced the apoptosis of p53 positive HCT-116 cells with G2/M phase arrest. Treatment with Baekduong-tang led to increased expression and phosphorylation of p53 and decreased expression of CDK2 and CDK6 in HCT-116 cells. It also activated caspase-3 through caspase-10 and caspase-9 activation. Finally, Baekduong-tang induced production $H_2O_2$, superoxide anion ($O_2^-$) and NO and modulated proteins expression including SOD, NOS, Bax and Bcl-2. Conclusions : These results indicate Baekduong-tang induces apoptotic death of HCT-116 cells through G2/M phase arrest and disturbance of intracellular redox status in a p53-dependent manner.

HCT116 대장암 세포에서 Akt-p53 신호경로를 통한 커큐민과 EGCG의 apoptosis 효과

Song-Yi Park(박송이),Sol-Hwa Lee(이솔화),Ock-Jin Park(박옥진),Young-Min Kim(김영민) 한국생명과학회 2011 생명과학회지 Vol.21 No.1

식품에서 추출한 파이토케미컬은 여러 암종에서 암세포의 증식억제와 apoptosis를 유도한다. 최근에 이러한 파이토케미컬의 세포 내 신호전달 기작에 관한 관심이 높아지고 있으며, 본 연구에서는 파이토케미컬의 일종인 커큐민과 EGCG를 HCT116 대장암세포에 처리함으로써 암세포의 증식억제와 apoptosis 유도 효과를 알아보고, 암세포의 증식에 관여하는 Akt의 활성과 종양 억제유전자인 p53의 신호경로를 규명하고자 하였다. 그 결과, 커큐민과 EGCG를 처리했을 때 HCT116 세포의 증식이 억제되었고, 암세포에서 apoptosis 효과가 나타남을 확인하였다. 동일한 조건에서 Western blotting을 실시했을 때 Akt의 활성은 감소하였으며 p53의 발현은 증가하였다. 또한 Akt의 저해제인 LY294002를 처리했을 때 암세포의 증식이 더욱 강하게 억제되었으며, p53의 발현은 더욱 강하게 증가하는 것으로 나타났다. 따라서 HCT116 세포에서 커큐민과 EGCG 처리에 의한 암세포의 증식 억제 및 apoptosis는 p53의 발현이 증가함에 따라 유도되며, 이러한 p53의 발현 증가는 Akt 신호경로를 저해함으로써 일어난다는 것을 확인하였다. p53 is tumor suppressor gene that regulates apoptosis such as caspase-dependent and p21-mediated signaling pathways. PI3K/Akt is known to be over-activated in cancer cells. Akt activates many survival-related signals such as mTOR and COX-2. Inactivation of Akt would result in non-inhibition of p53 as well as induced apoptosis. In this study, we showed that curcumin and EGCG activate p53 via inhibition of the Akt signaling pathway. Treatments using curcumin and EGCG in different concentrations for 24 hr and 48 hr inhibited proliferation of HCT116 colon cancer cells and increased apoptotic cell death. Also, our data showed that curcumin and EGCG increased the p53 expression and decreased the p-Akt. Treatment of LY294002 (Akt inhibitor) resulted in decreased cell proliferation of cancer cells, while LY294002 treated with curcumin or EGCG showed a greater decrease of cell proliferation. In addition, inhibition of Akt induced p53 activation in HCT116 colon cancer cells. These results suggest that curcumin and EGCG induce apoptosis by inhibiting Akt and increase p53 in HCT116 colon cancer cells.

HCT116 대장암세포에서 AKT/mTOR/GSK-3β 신호경로 조절을 통한 벌 사상자 추출물(CME)의 apoptosis 및 cell cycle arrest 효과

임은경(Eun Gyeong Lim),김근태(Guen Tae Kim),김보민(Bo Min Kim),김은지(Eun Ji Kim),하성호(Sung Ho Ha),김상용(Sang-Yong Kim),김영민(Young Min Kim) 한국생명과학회 2016 생명과학회지 Vol.26 No.6

벌 사상자[Cnidium monnieri (L.) Cusson]는 중국과 한국에 분포하는 일년생 초본으로, 화농성피부염 및 여성의 생식기 질환의 치료에 널리 사용되고 있다. 이 외에도 면역기능개선과 천식 등에 대한 효과는 보고된 바 있으나 아직까지 암과 관련된 연구는 많이 이루어지지 않았다. 이에 따라 본 연구에서는 인간 대장암 세포인 HCT116 세포주에서 벌 사상자 에탄올 추출물(CME)의 apoptosis 및 세포주기정지 유도 효과에 대하여 알아보고자 하였으며, 이러한 효과가 AKT/mTOR/GSK-3β 신호경로의 조절을 통하여 이루어지는지 확인하고자 하였다. MTT assay와 LDH assay 결과, 벌 사상자 에탄올 추출물에 의하여 HCT116 세포의 세포생존율이 감소하였으며, 세포독성효과가 나타났다. 또한 벌 사상자 에탄올 추출물의 농도의존적으로 apoptotic body의 수와 apoptosis 비율이 증가하였으며, G1기에서 세포주기정지 유도 효과가 관찰되었다. 세포의 성장과 증식 및 분열에 관련된 단백질인 Akt는 mTOR, p53, GSK-3β와 같은 신호단백질들의 발현을 조절하는 것으로 보고되었다. 벌 사상자 에탄올 추출물을 처리하였을 때, Akt와 mTOR 단백질의 인산화가 저해되었으며, 이에 따라 하위 신호조절 단백질인 GSK-3β, Bcl-2 family, Caspase-3, PARP의 발현이 조절되었다. 또한 Akt와 GSK-3β, mTOR 저해제 처리를 통하여 CME에 의한 apoptosis 효과가 AKT/mTOR/GSK-3β 신호경로를 통하여 이루어지는 것을 확인하였다. 결론적으로, 본 연구를 통하여HCT116 대장암 세포주에서 벌 사상자 에탄올 추출물이 암세포의 apoptosis 및 세포주기정지 유도에 효과적임을 확인하였다. The Cnidium monnieri (L.) Cusson is an annual plant distributed in China and Korea. The fruit of C. monnieri is used as a medicinal herb that is effective for the treatment of carbuncle and pain in female genitalia. However, the anti-cancer effects of CME have not yet been reported. In this study, we assessed the apoptotic effects and cell cycle arrest effects of ethanol extracts from C. monnieri on HCT116 colon cancer cells. The results of an MTT assay and LDH assay demonstrated a decrease in cell viability and the cytotoxic effects of CME. In addition, the number of apoptotic body and the apoptotic rate were increased in a dose-dependent manner through Hoechst 33342 staining and Annexin V-PI double staining. In addition, cell cycle arrest occurred at the G1 phase by CME. Protein kinase B (Akt) plays an important role in cancer cell survival, growth, and division. Akt down-regulates apoptosis-mediated proteins, such as mammalian target of rapamycin (mTOR), p53, and Glycogen Synthase kinase-3β (GSK-3β). CME could regulate the expression levels of p-Akt, p-mTOR, p-GSK-3β, Bcl-2 family members, caspase-3, and PARP. Furthermore, treatment with CME, LY294002 (PI3K/Akt inhibitor), BIO (GSK-3β inhibitor), and Rapamycin (mTOR inhibitor) showed that apoptotic effects occurred through the regulation of the AKT/mTOR/GSK-3β signaling pathway. Our results demonstrated CME could induce apoptosis and cell cycle arrest in HCT116 colon cancer cells.

Sequential administration of camptothecin sensitizes human colon cancer HCT116 cells to paclitaxel via p21^(Cip1/WAF1)

유정민,김윤진,이성재,김상훈 한국통합생물학회 2011 Animal cells and systems Vol.15 No.1

Colorectal cancer is the third leading cause of cancer-related death in Western countries. Chemotherapeutic agents with different mechanisms of action have shown an increase in cure rates. In the present study, we investigated the effect of a combination of low concentration of paclitaxel (taxol, 5 nM) and topoisomerase 1 inhibitor camptothecin (CPT) on HCT116 colon cancer cells. Although the viability of cells treated with taxol alone was similar to that of control cells, sequential treatment with taxol and CPT exhibited high cytotoxicity. However, the opposite sequence of treatment did not exert cytotoxic effects on HCT116 cells. This enhanced cytotoxicity of the sequential combination therapy was the result of mitotic arrest, which increased the level of p21^(Cip1/WAF1) through the p38 mitogen-activated protein kinase (MAPK) pathway. Knockdown by p21^(Cip1/WAF1) siRNA or treatment with a p38 inhibitor reduced the viability of cells sequentially exposed to taxol and CPT. Taken together, a low taxol concentration in combination with CPT induced mitotic arrest in HCT116 cells, leading to synergistic cell death through enhanced expression of p21^(Cip1/WAF1) and p38 MAPK pathway. Therefore, taxol could play a role as a sensitizer of CPT in colon cancer cells.

Sequential administration of camptothecin sensitizes human colon cancer HCT116 cells to paclitaxel via $p21^{Cip1/WAF1}$

Yoo, Jung-Min,Kim, Yun-Jin,Lee, Sung-Jae,Kim, Sang-Hoon The Korean Society for Integrative Biology 2011 Animal cells and systems Vol.15 No.1

Colorectal cancer is the third leading cause of cancer-related death in Western countries. Chemotherapeutic agents with different mechanisms of action have shown an increase in cure rates. In the present study, we investigated the effect of a combination of low concentration of paclitaxel (taxol, 5 nM) and topoisomerase 1 inhibitor camptothecin (CPT) on HCT116 colon cancer cells. Although the viability of cells treated with taxol alone was similar to that of control cells, sequential treatment with taxol and CPT exhibited high cytotoxicity. However, the opposite sequence of treatment did not exert cytotoxic effects on HCT116 cells. This enhanced cytotoxicity of the sequential combination therapy was the result of mitotic arrest, which increased the level of $p21^{Cip1/WAF1}$ through the p38 mitogen-activated protein kinase (MAPK) pathway. Knockdown by $p21^{Cip1/WAF1}$ siRNA or treatment with a p38 inhibitor reduced the viability of cells sequentially exposed to taxol and CPT. Taken together, a low taxol concentration in combination with CPT induced mitotic arrest in HCT116 cells, leading to synergistic cell death through enhanced expression of $p21^{Cip1/WAF1}$ and p38 MAPK pathway. Therefore, taxol could playa role as a sensitizer of CPT in colon cancer cells.

인체 대장암 세포주 HCT 116와 HT-29에서 아팝토시스 세포사멸과 자가소화작용의 이중유도에 의한 녹나무 에탄올 추출물의 mTOR 신호경로를 통한 항암효과

남건희,조경조,박예슬,곽혜원,위지향,김주환,김지훈,김영민 한국생물공학회 2019 KSBB Journal Vol.34 No.2

Cinnamomum camphora is known as a tree thathas been used in traditional medicine and food. However, itsbiological activities in cancer have not yet been clearly investigated. In this study, we investigate the anti-cancer effects ofethanol extract of Cinnamomum camphora on two kinds ofhuman colon cancer cell lines (HCT 116 and HT-29). In previousstudy, about 50% of cancer cells have p53 mutations. Therefore, two kinds of cancer cells with different p53 genemutation status are able to verify the effects of cancer moreaccurately. it is conducted several experiments to prove theeffects of anti-cancer through apoptosis and autophagy. It isconfirmed the anti-proliferative activity of Cinnamomumcamphora ethanol extract through WST-1 assay. Cancer cellmigration rate is detected by cell invasion assay and scratchedwound healing assay. Also, the expression of apoptosis andautophagy related proteins are assessed by western blot. Ourdata reveal that Cinnamomum camphora ethanol extractinhibits the cancer cell growth of both p53 gene wild typeHCT 116 human colon cancer cells and p53 gene mutationtype HT-29 human colon cancer cells. Moreover, it confirmsthat CCD841 Human colon normal cells are not affected byCinnamomum camphora ethanol extract. Cinnamomum camphoraethanol extract regulated expression of apoptotic andautophagic proteins in both cancer cells. Then, it is demonstratedthat apoptosis and autophagy are induced through themTOR pathway when treated with rapamycin (mTOR inhibitor). Taken together, these results show that Cinnamomumcamphora ethanol extract has considerable potential as chemotherapeuticsubstance.