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      Antitumor Efficacy of the Herbal Recipe Benja Amarit against Highly Invasive Cholangiocarcinoma by Inducing Apoptosis both In Vitro and In Vivo

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          Abstract

          Thailand is the country with highest incidence and prevalence of cholangiocarcinoma (CCA) in the world. Due to the frequently late diagnosis that is associated with this disease, most CCA patients are prescribed chemotherapy as a form of treatment. However, CCA is able to resist the presently available chemotherapy, so to the prognosis of this disease is still very poor. In this study, we investigated the anticancer potential of a Thai herbal recipe, Benja Amarit (BJA) against CCA and the relevant mechanisms of action that are involved. We found that BJA inhibited CCA cell viability in a dose-dependent manner, especially in highly invasive KKU-213 cells. The extract induced mitochondrial- and caspase-dependent apoptosis in CCA cells by regulating the nuclear factor-κB (NF-κB) signaling pathway. BJA also triggered autophagy in CCA cells. Nonetheless, the inhibition of autophagy enhanced BJA-induced CCA cell death via apoptosis. An in vivo xenograft model revealed the growth-inhibiting and death-inducing effects of BJA against CCA by targeting apoptosis. However, general toxicity to blood cells, kidneys and the liver, as well as changes in body weight, did not appear. Our findings suggest that the herbal recipe BJA might be used as a potentially new and effective treatment for cholangiocarcinoma patients.

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          The role of autophagy in cancer development and response to therapy.

          Autophagy is a process in which subcellular membranes undergo dynamic morphological changes that lead to the degradation of cellular proteins and cytoplasmic organelles. This process is an important cellular response to stress or starvation. Many studies have shed light on the importance of autophagy in cancer, but it is still unclear whether autophagy suppresses tumorigenesis or provides cancer cells with a rescue mechanism under unfavourable conditions. What is the present state of our knowledge about the role of autophagy in cancer development, and in response to therapy? And how can the autophagic process be manipulated to improve anticancer therapeutics?
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            The Roles of Autophagy in Cancer

            Autophagy is an intracellular degradative process that occurs under several stressful conditions, including organelle damage, the presence of abnormal proteins, and nutrient deprivation. The mechanism of autophagy initiates the formation of autophagosomes that capture degraded components and then fuse with lysosomes to recycle these components. The modulation of autophagy plays dual roles in tumor suppression and promotion in many cancers. In addition, autophagy regulates the properties of cancer stem-cells by contributing to the maintenance of stemness, the induction of recurrence, and the development of resistance to anticancer reagents. Although some autophagy modulators, such as rapamycin and chloroquine, are used to regulate autophagy in anticancer therapy, since this process also plays roles in both tumor suppression and promotion, the precise mechanism of autophagy in cancer requires further study. In this review, we will summarize the mechanism of autophagy under stressful conditions and its roles in tumor suppression and promotion in cancer and in cancer stem-cells. Furthermore, we discuss how autophagy is a promising potential therapeutic target in cancer treatment.
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              Semi-quantitative Determination of Protein Expression Using Immunohistochemistry Staining and Analysis: An Integrated Protocol

              Semi-quantitative IHC is a powerful method for investigating protein expression and localization within tissues. The semi-quantitative immunohistochemistry (IHC) involves using software such as free software ImageJ Fiji to conduct deconvolution and downstream analysis. Currently, there is lack of an integrated protocol that includes a detailed procedure of how to measure or compare protein expression. Publications that use semi-quantification methods to quantify protein expression often don’t provide enough details in their methods section, which makes it difficult for the reader to reproduce their data. The current protocol for the first time provides a detailed, step-by-step instruction of conducting semi-quantitative analysis of IHC images using ImageJ Fiji software so that researchers would be able to follow this single protocol to conduct their research. The protocol uses semi-quantitative IHC of organic anion transporting polypeptide (OATP1B1) as an example, and gives detailed steps on how to deconvolute IHC images stained with hematoxylin and 3,3’-diaminobenzidine (DAB) and how to quantify their expression using ImageJ Fiji. The protocol includes clear steps for a reader so that this method can be applied to many different proteins. We anticipate this method will provide a practical guidance to the reader and make semi-quantification of proteins an easier task to include in publications.
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                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                07 August 2020
                August 2020
                : 21
                : 16
                : 5669
                Affiliations
                [1 ]Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand; rittibet_ya@ 123456cmu.ac.th (R.Y.); subhawat_s@ 123456cmu.ac.th (S.S.); juthathip_p@ 123456cmu.ac.th (J.P.)
                [2 ]Department of Pathology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand; nlertpra@ 123456hotmail.com
                [3 ]Center for Research and Development of Herbal Health Products (CRD-HHP), Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand; bungorn@ 123456kku.ac.th
                [4 ]Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand
                Author notes
                [* ]Correspondence: ratana.b@ 123456cmu.ac.th ; Tel.: +66-53-935-325; Fax: +66-53-894-031
                Author information
                https://orcid.org/0000-0002-5157-7614
                Article
                ijms-21-05669
                10.3390/ijms21165669
                7460969
                32784671
                1957770a-9d09-4abe-86ce-a423acd0df74
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 21 July 2020
                : 03 August 2020
                Categories
                Article

                Molecular biology
                benja amarit,bja,herbal recipe,cholangiocarcinoma,liver cancer,apoptosis,autophagy,in vitro,in vivo

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