구강 편평세포암종에서의 암줄기세포 이론과 최신 지견

김덕훈(Deok-Hun Kim),윤준용(Jun-Yong Yun),이주현(Ju-Hyun Lee),김성민(Soung-Min Kim),명 훈(Hoon Myoung) 대한구강악안면외과학회 2011 대한구강악안면외과학회지 Vol.37 No.2

Cancer stem cells have stem cell-like features, such as the ability for self-renewal and differentiation but show unlimited growth because they have the lost normal regulation of cell growth. Cancer stem cells and normal stem cells have similar features. They show high motility, diversity of progeny, robust proliferative potential, association with blood vessels, immature expression profiles, nestin expression, epidermal growth factor (EGF)-receptor expression, phosphatase and tensin homolog (PTEN) expression, hedgehog pathway activity, telomerase activity, and Wnt pathway activity. On the other hand, with cancer cells, some of these signaling pathways are abnormally modified. In 1875, Cohnheim suggested the concept of cancer stem cells. Recently, evidence for the existence of cancer stem cells was identified. In 1994, the cancer stem cells’specific cell surface marker for leukemia was identified. Since then, other specific cell surface markers for cancer stem cells in solid tumors (e.g. breast and colon cancer) have been identified. In oral cancer, studies on cancer stem cells have been performed mainly with squamous cell carcinomas. Oral cancer specific cell surface markers, which are genes strongly expressed in oral cancer and cancer stem cell specific side populations, have been identified. Cancer stem cells are resistant to radiotherapy and chemotherapy. Therefore, to eliminate malignant tumors efficiently and reduce the recurrence rate, therapy targeting cancer stem cells needs to be performed. Currently, studies targeting the cancer stem cells’specific signaling pathways, telomerase and tumor vasculatures are being done.

Functionalizing Liposomes with Dual Aptamers for Targeting of Breast Cancer Cells and Cancer Stem Cells

Hee-Bin Park,Ji-Eun You,Pyung-Hwan Kim,Keun-Sik Kim 대한의생명과학회 2021 Biomedical Science Letters Vol.27 No.1

Cancer stem cells, which are known to drive tumor formation and maintenance, are a major obstacle in the effective treatment of various types of cancer. Trans-membrane glycoprotein mucin 1 antigen and cell surface glycogen CD44 antigen are well-known surface markers of breast cancer cells and breast cancer stem cells, respectively. To effectively treat cancer cells and cancer stem cells, we developed a new drug-encapsulating liposome conjugated with dual-DNA aptamers specific to the surface markers of breast cancer cells and their cancer stem cells. These two aptamer (Apt)-targeted liposomes, which were prepared to encapsulate doxorubicin (Dox), were named "Dual-Apt-Dox". Dual-Apt-Dox is significantly more cytotoxic to both cancer stem cells and cancer cells compared to liposomes lacking the aptamers. Furthermore, we demonstrated the inhibitory efficacy of Dual-Apt-Dox against the experimental lung metastasis of breast cancer stem cells and cancer cells in athymic nude mice. We also showed the potent antitumor effects of dualaptamer-conjugated liposome systems by targeting cancer cells as well as cancer stem cells. Thus, our data indicate that dual-aptamer-conjugated liposome systems can prove to be effective drug delivery vehicles for breast cancer therapy.

유방암 줄기세포 개념 및 제한점

김종빈,안정신,임우성,문병인,Kim, Jong Bin,An, Jeong Shin,Lim, Woosung,Moon, Byung-In 제주대학교 의과학연구소 2018 The Journal of Medicine and Life Science Vol.15 No.2

Cancer, a leading mortality disease following cardiovascular disease worldwide, has high incidence as one out of every four adults in Korea. It was known to be caused by several reasons including somatic mutation, activation of oncogene and chromosome aneuploidy. Cancer cells show a faster growth rate and have metastatic and heterogeneous cell populations compared to normal cells. Cancer stem cells, the most invested field in cancer biology, is a theory to explain heterogeneous cell populations of cancer cells among several characteristics of cancer cells, which is providing the theoretical background for incidence of cancer and treatment failure by drug resistance. Cancer stem cells initially explain heterogeneous cell populations of cancer cells based on the same markers of normal stem cells in cancer, in which only cancer stem cells showed heterogeneity of cancer cells and tumor initiating ability of leukemia. Based on these results, cancer stem cells were reported in various solid cancers such as breast cancer, liver cancer, and lung cancer. Breast cancer stem cells were first reported in solid cancer which had tumor initiating ability and further identified as anti-cancer drug resistance. There were several identification methods in breast cancer stem cells such as specific surface markers and culture methods. The discovery of cancer stem cells not only explains heterogeneity of cancer cells, but it also provides theoretical background for targeting cancer stem cells to complete elimination of cancer cells. Many institutes have been developing new anticancer drugs targeting cancer stem cells, but there have not been noticeable results yet. Many researchers also reported a necessity for improvement of current concepts and methods of research on cancer stem cells. Herein, we discuss the limitations and the perspectives of breast cancer stem cells based on the current concept and history.

Correlation analysis of cancer stem cell marker CD133 and human endogenous retrovirus (HERV)-K env in SKOV3 ovarian cancer cells

Kim Do-Ye,Kim Heungyeol,Ko Eun-Ji,Koh Suk Bong,Kim Hongbae,Lee Ji Young,Lee Chul Min,Eo Wan Kyu,Kim Ki Hyung,Cha Hee-Jae 한국유전학회 2024 Genes & Genomics Vol.46 No.4

Background Human endogenous retrovirus (HERV)-K is a type of retrovirus that is present in the human genome, and its expression is usually silenced in healthy tissues. The precise mechanism by which HERV-K env influences cancer stemness is not fully understood, but it has been suggested that HERV-K env may activate various signaling pathways that promote stemness traits in cancer cells. Objective To establish the connection between HERV-K env expression and cancer stemness in ovarian cancer cells, we carried out correlation analyses between HERV-K env and the cancer stem cell (CSC) marker known as the cluster of differentiation 133 (CD133) gene in SKOV3 ovarian cancer cells. Method To perform correlation analysis between HERV-K env and CSCs, ovarian cancer cells were cultured in a medium designed for cancer stem cell induction. The expression of HERV-K env and CD133 genes was verified using quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Additionally, the expression of stemness-related markers, such as OCT-4 and Nanog, was also confirmed using RT-qPCR. Results In the stem cell induction medium, the number of tumorsphere-type SKOV3 cells increased, and the expression of CD133 and HERV-K env genes was up-regulated. Additionally, other stemness-related markers like OCT-4 and Nanog also exhibited increased expression when cultured in the cancer stem cell induction medium. However, when HERV-K env knockout (KO) SKOV3 cells were cultured in the same cancer stem cell induction medium, there was a significant decrease in the number of tumorsphere-type cells compared to mock SKOV3 cells subjected to the same conditions. Furthermore, the expression of CD133, Nanog, and OCT-4 did not show a significant increase in HERV-K env KO SKOV3 cells compared to mock SKOV3 cells cultured in the same cancer stem cell induction medium. Conclusion These findings indicate that the expression of HERV-K env increased in SKOV3 cells when cultured in cancer stem cell induction media, and cancer stem cell induction was inhibited by KO of HERV-K env in SKOV3 cells. These results suggest a strong association between HERV-K env and stemness in SKOV3 ovarian cancer cells. Background Human endogenous retrovirus (HERV)-K is a type of retrovirus that is present in the human genome, and its expression is usually silenced in healthy tissues. The precise mechanism by which HERV-K env influences cancer stemness is not fully understood, but it has been suggested that HERV-K env may activate various signaling pathways that promote stemness traits in cancer cells. Objective To establish the connection between HERV-K env expression and cancer stemness in ovarian cancer cells, we carried out correlation analyses between HERV-K env and the cancer stem cell (CSC) marker known as the cluster of differentiation 133 (CD133) gene in SKOV3 ovarian cancer cells. Method To perform correlation analysis between HERV-K env and CSCs, ovarian cancer cells were cultured in a medium designed for cancer stem cell induction. The expression of HERV-K env and CD133 genes was verified using quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Additionally, the expression of stemness-related markers, such as OCT-4 and Nanog, was also confirmed using RT-qPCR. Results In the stem cell induction medium, the number of tumorsphere-type SKOV3 cells increased, and the expression of CD133 and HERV-K env genes was up-regulated. Additionally, other stemness-related markers like OCT-4 and Nanog also exhibited increased expression when cultured in the cancer stem cell induction medium. However, when HERV-K env knockout (KO) SKOV3 cells were cultured in the same cancer stem cell induction medium, there was a significant decrease in the number of tumorsphere-type cells compared to mock SKOV3 cells subjected to the same conditions. Furthermore, the expression of CD133, Nanog, and OCT-4 did not show a significant increase in HERV-K env KO SKOV3 cells compared to mock SKOV3 cells cultured in the same cancer stem cell induction medium. Conclusion These findings indicate that the expression of HERV-K env increased in SKOV3 cells when cultured in cancer stem cell induction media, and cancer stem cell induction was inhibited by KO of HERV-K env in SKOV3 cells. These results suggest a strong association between HERV-K env and stemness in SKOV3 ovarian cancer cells.

A mutual activation loop between breast cancer cells and myeloid-derived suppressor cells facilitates spontaneous metastasis through IL-6 trans-signaling in a murine model

Oh, Keunhee,Lee, Ok-Young,Shon, Suh Youn,Nam, Onyou,Ryu, Po Mee,Seo, Myung Won,Lee, Dong-Sup BioMed Central 2013 Breast cancer research Vol.15 No.5

<P><B>Introduction</B></P><P>Tumor cell interactions with the microenvironment, especially those of bone-marrow-derived myeloid cells, are important in various aspects of tumor metastasis. Myeloid-derived suppressor cells (MDSCs) have been suggested to constitute tumor-favoring microenvironments. In this study, we elucidated a novel mechanism by which the MDSCs can mediate spontaneous distant metastasis of breast cancer cells.</P><P><B>Methods</B></P><P>Murine breast cancer cells, 4T1 and EMT6, were orthotopically grafted into the mammary fat pads of syngeneic BALB/c mice. CD11b<SUP>+</SUP>Gr-1<SUP>+ </SUP>MDSCs in the spleen, liver, lung and primary tumor mass were analyzed. To evaluate the role of MDSCs in the distant metastasis, MDSCs were depleted or reconstituted in tumor-bearing mice. To evaluate whether MDSCs in the metastasizing tumor microenvironment affect breast cancer cell behavior, MDSCs and cancer cells were co-cultivated. To investigate the role of MDSCs in <I>in vivo </I>metastasis, we blocked the interactions between MDSCs and cancer cells.</P><P><B>Results</B></P><P>Using a murine breast cancer cell model, we showed that murine breast cancer cells with high IL-6 expression recruited more MDSCs and that the metastasizing capacity of cancer cells paralleled MDSC recruitment in tumor-bearing mice. Metastasizing, but not non-metastasizing, tumor-derived factors induced MDSCs to increase IL-6 production and full activation of recruited MDSCs occurred in the primary tumor site and metastatic organ in the vicinity of metastasizing cancer cells, but not in lymphoid organs. In addition, tumor-expanded MDSCs expressed Adam-family proteases, which facilitated shedding of IL-6 receptor, thereby contributing to breast cancer cell invasiveness and distant metastasis through IL-6 trans-signaling. The critical role of IL-6 trans-signaling was confirmed in both the afferent and efferent pathways of metastasis.</P><P><B>Conclusion</B></P><P>In this study, we showed that metastasizing cancer cells induced higher MDSCs infiltration and prompted them to secret exaggerated IL-6 as well as soluble IL-6Rα, which, in turn, triggered a persistent increase of pSTAT3 in tumor cells. This potential tumor-MDSC axis involving IL-6 trans-signaling directly affected breast cancer cell aggressiveness, leading to spontaneous metastasis.</P>

Cytokeratin 19 <i>(KRT19)</i> has a Role in the Reprogramming of Cancer Stem Cell-Like Cells to Less Aggressive and More Drug-Sensitive Cells

Saha, Subbroto Kumar,Kim, Kyeongseok,Yang, Gwang-Mo,Choi, Hye Yeon,Cho, Ssang-Goo MDPI AG 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.5

<P>Cytokeratin 19 (<I>KRT19</I>) is a cytoplasmic intermediate filament protein, which is responsible for structural rigidity and multipurpose scaffolds. In several cancers, <I>KRT19</I> is overexpressed and may play a crucial role in tumorigenic transformation. In our previous study, we revealed the role of <I>KRT19</I> as signaling component which mediated Wnt/NOTCH crosstalk through NUMB transcription in breast cancer. Here, we investigated the function of <I>KRT19</I> in cancer reprogramming and drug resistance in breast cancer cells. We found that expression of <I>KRT19</I> was attenuated in several patients-derived breast cancer tissues and patients with a low expression of <I>KRT19</I> were significantly correlated with poor prognosis in breast cancer patients. Consistently, highly aggressive and drug-resistant breast cancer patient-derived cancer stem cell-like cells (konkuk university-cancer stem cell-like cell (KU-CSLCs)) displayed higher expression of cancer stem cell (CSC) markers, including <I>ALDH1</I>, <I>CXCR4</I>, and <I>CD133</I>, but a much lower expression of <I>KRT19</I> than that is seen in highly aggressive triple negative breast cancer MDA-MB231 cells. Moreover, we revealed that the knockdown of <I>KRT19</I> in MDA-MB231 cells led to an enhancement of cancer properties, such as cell proliferation, sphere formation, migration, and drug resistance, while the overexpression of <I>KRT19</I> in KU-CSLCs resulted in the significant attenuation of cancer properties. <I>KRT19</I> regulated cancer stem cell reprogramming by modulating the expression of cancer stem cell markers (<I>ALDH1</I>, <I>CXCR4</I>, and <I>CD133</I>), as well as the phosphorylation of Src and GSK3β (Tyr216). Therefore, our data may imply that the modulation of <I>KRT19</I> expression could be involved in cancer stem cell reprogramming and drug sensitivity, which might have clinical implications for cancer or cancer stem cell treatment. </P>

Histopathological Evidence for the Existence of Primary Liver Progenitor Cell Cancer: Insight from Cancer Stem Cell Pathobiology

( Cheng-maw Ho ),( Shu-li Ho ),( Chia-tung Shun ),( Po-huang Lee ),( Ya-hui Chen ),( Chin-sung Chien ),( Hui-ling Chen ),( Rey-heng Hu ) 대한간학회 2017 춘·추계 학술대회 (KASL) Vol.2017 No.1

Aims: Primary liver progenitor cell cancer is a rare disease entity without definite evidence and characterization. Current nomenclature of primary liver cancer with prominent progenitor features is not comprehensive. This study was aimed to investigate the existence of this kind of primary liver cancer and characterize it immunohistopathologically based on the emerging understanding of cancer stem cell pathobiology. Methods: Surgical specimens from primary liver cancer which posed diagnostic difficulty fitting within current WHO classification of combined hepatocellular-cholangiocellular carcinoma with stem-cell features according to the growth morphology and its suggested immunohistochemical features, were stained with antibodies against well-defined markers of progenitor cells, stemness, and differentiation toward hepatocytes or cholangiocytes. Comparative interpretation of images was processed considering the histological morphology and characteristic markers. Results: The primary liver cancer consisted of CD24+ cancer progenitor cells and CD90+ mesenchymal stromal cells, which were intimately mixed. CD24+ cancer cells demonstrated bi-directional trends of differentiation: bile ductule transformation (cytokeratin 19+, epithelial cell adhesion molecule [EpCAM]+, neural cell adhesion molecule [NCAM]+, CD133+, and delta-like 1 homolog [DLK1]+); and trabecular or nested cell clusters toward hepatic lineage (hepatocyte nuclear factor-4 alpha [HNF-4α]+, Hep Par1+ and negative for CK19, EpCAM, CD133, and DLK1). Moderate lymphocyte (mostly CD4+ and CD8+ T cells) infiltrated in the CD90+ cancer- associated stroma. Conclusions: We provided the corroboration that liver progenitor cells can form primary liver cancer, not just presented as few side population of cancer stem cells. Its existence might pose significance for future stem cell therapeutic intervention targeting liver diseases, albeit the disease is rare.

Dehydroabietic acid inhibits the gastric cancer cell growth via induced apoptosis and cell cycle arrest

김원진,Kang Hyeon-Gu,김석준 대한독성 유전단백체 학회 2021 Molecular & cellular toxicology Vol.17 No.2

Background Gastric cancer is one of the serious malignant tumors with high incidence worldwide. The two commonly used treatment strategies for gastric cancer include chemotherapy and radiation therapy. Conventional anticancer drugs have limited effectiveness. Therefore, it is important to explore new anticancer drugs. Dehydroabietic acid (DAA) is known to have anti-bacterial, anti-inflammatory, and anticancer effects. However, there are fewer reports on the effect of DAA on human gastric cancer cell lines. Objective We investigated the gastric cell growth inhibitory effects of DAA on human gastric cancer cell lines. Gastric cancer cell proliferation, apoptosis and cell cycle arrest assay were detected by WST assay, crystal violet staining assay, Flow cytometry, RT-PCR and western blot analysis. Results These results showed that as the concentration of DAA increased, the proliferation inhibitory effect appeared in gastric cancer cells. In addition, DAA induced sub-G1 phase accumulation and G1 cell cycle arrest, and subsequently induced apoptosis in AGS and YCC-2 cells. In addition, the expression of the pro-apoptotic Bax was upregulated, and that of the anti-apoptotic Bcl-2 was downregulated in DAA-treated AGS and YCC-2 cells. Moreover, DAA induced the cleavage of caspase-3 and PARP. Conclusion These results suggest that DAA induces cell death through mitochondria-mediated apoptosis and G1 cell cycle arrest. Therefore, our results indicate the anticancer and therapeutic potential of DAA for treatment of human gastric cancer. Background Gastric cancer is one of the serious malignant tumors with high incidence worldwide. The two commonly used treatment strategies for gastric cancer include chemotherapy and radiation therapy. Conventional anticancer drugs have limited effectiveness. Therefore, it is important to explore new anticancer drugs. Dehydroabietic acid (DAA) is known to have anti-bacterial, anti-inflammatory, and anticancer effects. However, there are fewer reports on the effect of DAA on human gastric cancer cell lines. Objective We investigated the gastric cell growth inhibitory effects of DAA on human gastric cancer cell lines. Gastric cancer cell proliferation, apoptosis and cell cycle arrest assay were detected by WST assay, crystal violet staining assay, Flow cytometry, RT-PCR and western blot analysis. Results These results showed that as the concentration of DAA increased, the proliferation inhibitory effect appeared in gastric cancer cells. In addition, DAA induced sub-G1 phase accumulation and G1 cell cycle arrest, and subsequently induced apoptosis in AGS and YCC-2 cells. In addition, the expression of the pro-apoptotic Bax was upregulated, and that of the anti-apoptotic Bcl-2 was downregulated in DAA-treated AGS and YCC-2 cells. Moreover, DAA induced the cleavage of caspase-3 and PARP. Conclusion These results suggest that DAA induces cell death through mitochondria-mediated apoptosis and G1 cell cycle arrest. Therefore, our results indicate the anticancer and therapeutic potential of DAA for treatment of human gastric cancer.

人繼代 암세포주 성장 저해에 미치는 영지의 항암효과

오환용(Whan Young Oh),고승오(Seung O Ko),신효근(Hyo Keun Shin),김오환(Oh Whan Kim) 대한구강악안면외과학회 1996 대한구강악안면외과학회지 Vol.22 No.3

The effective way of control cancers currently relies on early case finding and treatment but the best way is more fundamental prevention through inhibition of many stages of carcinogenesis. Since many cancers are related to environmental and life style factors, recently chemoprevention through micronutrients, nonnutrient plant substances, synthetic nutrient derivatives, and synthetic chemicals have been attracted interest of cancer researcher. Almost 40clinical chemoprevention trials are now in progress around world among which γcarotene and 13cis retinoic acid were proven to be effetive in many cancers including colon, lung, breast, skin, cervix and head and neck cancer. The main purpose of this study is to get a basic data for the development of chemopreventive agents in Korean Herbs. It has been reported that Ganoderma has a hypotensive effect through the inhibition of angiotensin converting enzyme, antiallergic and antiasthmatic effect through the inhibition of histamin release from the mast cells, in addition to an inhibitory effect of lipid peroxidation, and a potenting effect of the immune system. Attempts were made to see the anticancer effect of Ganoderma extracted with hot water a common preparative method in most herb medicine, employing human cancer cell lines such as K562 derived from erythroleukemia, Raji from lymphoma and Molt4 from blastogenic tumors. Apart from those tumor cell suspinsions derived from hematopoietic cells, the anticancer effect of Ganoderma was also investigated with other human solid tumors of liver, lung, kidney and stomach cancers. The anticancer effect of Ganodrma of three different preparations, that is, a naturally grown one, a cultivated one and a molded one during malstorage, was also compared. The results obtained from this study were as follows. 1. Inhibitory effect of cancer cell growth by Ganoderma in 5different human solid tumor cell lines such as KHOS/MP, SDK, HeLa, A549, HepG2 was 6-28% showing highest effect in HeLa and lowest effect in HepG2 among tested. 2. Native Ganoderma showed 7-28%, inhibitory effect of cancer cell growth in 5different human solid tumor cell lines tested, and cultivated Ganoderma showed 6-28%, indicating that native and cultivated one do not reveal the difference on the cancer cell inhibitory effect. Mal-stored Ganoderma with fungal contanimation showed only 0-12%, half value of cancer cell inhibitory effect. 3. On the contrary to the solid human cancer cell lines, cancer cell suspensions like K562 derived from chronic myelogenous leukemia showed 72% Molt4 derived from acute lymphoblastic leukemia showed 71%, and Raji derived from Burkitt lymphoma showed 58%, indicating that Ganoderma has more anticancer effect on cancer cell suspensions derived from hematopoietic system than solid tumors tested in this study. 4. In the dosage dependent study of Ganoderma together with 5-fluorouracil(5 FU) as referenfe drug, HeLa cell was most effective showing 30% of cancer cell cytotoxicity at the 1mg/ml, while 5FU showed 47% of cancer cell cytotoxicity at the same concentration. Above results revealed Ganoderma was more effective to the cancer cell suspensions derived from hematopoietic system than solid tumors. And cancer cell cytotoxicity is not almost different both in native and cultivated Ganoderma.

Proliferative and Inhibitory Activity of Siberian ginseng (Eleutherococcus senticosus) Extract on Cancer Cell Lines; A-549, XWLC-05, HCT-116, CNE and Beas-2b

Cichello, Simon Angelo,Yao, Qian,Dowell, Ashley,Leury, Brian,He, Xiao-Qiong Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.11

Siberian ginseng (Eleutherococcus senticosus) is used primarily as an adaptogen herb and also for its immune stimulant properties in Western herbal medicine. Another closely related species used in East Asian medicine systems i.e. Kampo, TCM (Manchuria, Korea, Japan and Ainu of Hokkaido) and also called Siberian ginseng (Acanthopanax senticosus) also displays immune-stimulant and anti-cancer properties. These may affect tumour growth and also provide an anti-fatigue effect for cancer patients, in particular for those suffering from lung cancer. There is some evidence that a carbohydrate in Siberian ginseng may possess not only immune stimulatory but also anti-tumour effects and also display other various anti-cancer properties. Our study aimed to determine the inhibitory and also proliferative effects of a methanol plant extract of Siberan ginseng (E. senticosus) on various cancer and normal cell lines including: A-549 (small cell lung cancer), XWLC-05 (Yunnan lung cancer cell line), CNE (human nasopharyngeal carcinoma cell line), HCT-116 (human colon cancer) and Beas-2b (human lung epithelial). These cell lines were treated with an extract from E. senticosus that was evaporated and reconstituted in DMSO. Treatment of A-549 (small cell lung cancer) cells with E. senticosus methanolic extract showed a concentration-dependent inhibitory trend from $12.5-50{\mu}g/mL$, and then a plateau, whereas at 12.5 and $25{\mu}g/mL$, there is a slight growth suppression in QBC-939 cells, but then a steady suppression from 50, 100 and $200{\mu}g/mL$. Further, in XWLC-05 (Yunnan lung cancer cell line), E. senticosus methanolic extract displayed an inhibitory effect which plateaued with increasing dosage. Next, in CNE (human nasopharyngeal carcinoma cell line) there was a dose dependent proliferative response, whereas in Beas-2 (human lung epithelial cell line), an inhibitory effect. Finally in colon cancer cell line (HCT-116) we observed an initially weak inhibitory effect and then plateau.