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Background: Indole-3-carbinol and its metabolic products are considered promising chemopreventive and anticancer agents. Previously we have shown that the indole-3-carbinol cyclic tetrameric derivative CTet induces autophagy and inhibits cell proliferation via inhibition of Akt activity and overexpression of p21/CDKN1A and GADD45A, in both estrogen receptor-positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cell lines. In the present study, we further characterize the autophagic response and investigate the mechanism through which CTet regulates these events.

Methodology/principal findings: Analysis of gene expression microarray data and subsequent confirmation by quantitative real-time PCR, showed that CTet is able to induce up-regulation of key signaling molecules involved in endoplasmic reticulum (ER) stress response (e.g. DDIT3/CHOP, CHAC1, ATF3, HSPA5/BiP/GRP78, CEBPB, ASNS) and autophagy (e.g. MAP1LC3B), in both MCF-7 and MDA-MB-231 cell lines. Moreover, the monitoring of Xbp-1 splicing confirmed the activation of IRE1/Xbp-1 ER stress response branch after CTet treatment. The role of autophagic processes (known to be induced by ER stress) was investigated further through ATG5 gene silencing and pharmacological inhibition of AVOs formation. CTet was shown to induce an autophagy-related cell death. Moreover, CTet-treated cells stained with Hoechst/PI revealed the presence of necrotic processes without evidence of apoptosis.

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Conclusions/significance: The ER stress response was identified as the main upstream molecular mechanism through which CTet acts in both hormone-responsive and triple-negative breast cancer cells. Because of its important role in cancer development, ER stress is a potential target in cancer therapy. The abiltiy of CTet to induce ER stress response and subsequently activate a death program in tumor cells confirms this molecule as a promising anticancer agent.

The CBSE CTET Final Answer Key 2023 for both Paper 1 and Paper 2 will be released together on www.ctet.nic.in. The CTET Final Answer Key will be made available online for all the candidates who have appeared in the CTET 2023 Exam. Over 29 lakh candidates applied for the CTET Exam, which was held on 20th August 2023 with 80% attendance. Out of the total candidates, 15,01,719 registered for paper 1 (classes 1-5) and 14,02,184 for paper 2 (classes 6-8). There will be no negative marking for incorrect answers in the CTET Aug-2023 Exam. Calculate your marks before the result with the help of the CTET Answer Key.

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Indole-3-carbinol and its metabolic products are considered promising chemopreventive and anticancer agents. Previously we have shown that the indole-3-carbinol cyclic tetrameric derivative CTet induces autophagy and inhibits cell proliferation via inhibition of Akt activity and overexpression of p21/CDKN1A and GADD45A, in both estrogen receptor-positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cell lines. In the present study, we further characterize the autophagic response and investigate the mechanism through which CTet regulates these events.

Analysis of gene expression microarray data and subsequent confirmation by quantitative real-time PCR, showed that CTet is able to induce up-regulation of key signaling molecules involved in endoplasmic reticulum (ER) stress response (e.g. DDIT3/CHOP, CHAC1, ATF3, HSPA5/BiP/GRP78, CEBPB, ASNS) and autophagy (e.g. MAP1LC3B), in both MCF-7 and MDA-MB-231 cell lines. Moreover, the monitoring of Xbp-1 splicing confirmed the activation of IRE1/Xbp-1 ER stress response branch after CTet treatment. The role of autophagic processes (known to be induced by ER stress) was investigated further through ATG5 gene silencing and pharmacological inhibition of AVOs formation. CTet was shown to induce an autophagy-related cell death. Moreover, CTet-treated cells stained with Hoechst/PI revealed the presence of necrotic processes without evidence of apoptosis.

The ER stress response was identified as the main upstream molecular mechanism through which CTet acts in both hormone-responsive and triple-negative breast cancer cells. Because of its important role in cancer development, ER stress is a potential target in cancer therapy. The abiltiy of CTet to induce ER stress response and subsequently activate a death program in tumor cells confirms this molecule as a promising anticancer agent.

Autophagy, a conserved cellular pathway activated in response to starvation and after treatment with some chemotherapeutic drugs, can also be induced by endoplasmic reticulum (ER) stress [3]. The ER stress is caused by perturbation of ER functions (i.e. protein synthesis/folding/post-translational modifications, biosynthesis of lipids and sterols, Ca2+ storage), and it is sensed by three ER-transmembrane transducers: ATF6, IRE1 and PERK [4], [5]. IRE1 and PERK are activated by phosphorylation, while ATF6 is translocated to the Golgi apparatus and cleaved by intramembrane proteolysis to release the transcriptionally active N-terminal domain. The activated ATF6 stimulates the expression of genes containing ER stress elements (ERSE-I, -II), UPR elements (UPRE), and cAMP response elements (CRE) in their promoters [4]. The activated IRE1 induces the unconventional splicing of X-box binding protein 1 (Xbp-1) mRNA [6]. In metazoans, a 26-nucleotide intron is spliced out, leading to a spliced form of Xbp-1 mRNA (sXbp-1) which encodes a highly active transcription factor belonging to the basic-leucine zipper (bZIP) family. The sXbp-1 protein induces the expression of several genes encoding ER chaperones (e.g. HSPA5/BiP/GRP78, ERdj4/DNAJB9) [7] and proteins involved in ER-associated protein degradation (ERAD) (e.g. HERPUD1, HRD1) [8], [9]. PERK (EIF2AK3) is a protein kinase which phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2), leading to global translation attenuation. At the same time, phosphorylated eIF2 induces selective translation of activating transcription factor 4 (ATF4). ATF4, in turn, induces the expression of several genes including amino acid transporters, chaperones, and C/EBP homologous protein (DDIT3/CHOP). Together, these three branches mitigate ER stress by reducing protein synthesis, facilitating protein degradation, and increasing production of chaperones. One consequence of ER stress is the accumulation of reactive oxygen species (ROS) that promotes a state of oxidative stress. PERK signaling also engages survival responses against oxidative stress by inducing the expression of genes involved in the oxidative stress response [10]. When ER stress is prolonged and it is not possible to recover the ER function, the apoptotic pathway is activated [5]. To repair tissue damage caused by cell death, the ER stress also induces an inflammatory response through the expression of several inflammatory cytokines (e.g. IL-6, IL-8) [11].

The ER stress has an important role in cancer development, therefore being a potential target in cancer therapy [12]. The artificial induction of ER stress response in tumor cells, which causes the activation of a death program, may be used in the development of anticancer drugs. Several anticancer compounds eliciting ER stress response have been described by Healy et al. [12].

This study was aimed to investigate the specific stress response pathways activated by CTet in estrogen receptor-positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cell lines, by looking for up-regulation/activation of key signaling molecules, and further characterization of the autophagic response. Induction of ER stress response, together with autophagy-related cell death were identified as major consequences of CTet treatment of the breast cancer cells tested.

Microarray analysis has been used to examine the transcriptional response elicited by CTet treatment in MCF-7 and MDA-MB-231 cell lines [2]. Complete microarray data are available in ArrayExpress database (accession number: E-MEXP-2989). In the present investigation a list of 116 genes significantly up-regulated in both cell lines after 24 h treatment with 6 M and 12 M CTet [2] was pruned by hand to delete duplicate genes or genes with no associated ontologies, obtaining a list of 92 up-regulated genes with known functions. Among these genes, a relatively large number of genes with roles in ER stress response and related functions, such as response to starvation and autophagy were identified by searching literature (Table 1).

One effect of ER stress is the activation of transcription factor Xbp-1 by unconventional splicing of its mRNA mediated by IRE1, leading to the elimination of a 26-nucleotide intron [6]. We determined whether the Xbp-1 transcript was subjected to this splicing in MCF-7 and MDA-MB-231 cells treated with CTet 12 M. The treated cells were harvested at 4 h, 8 h and 24 h, and Xbp-1 splicing was monitored as described in methods. For each time point, cells treated with tunicamycin were considered as positive control. In fact, tunicamycin is known to inhibit glycosylation of newly synthesized proteins, inducing ER stress and Xbp-1 mRNA splicing [14]. -Cyclodextrin-treated cells and DMSO-treated cells were used as negative controls for CTet and tunicamycin treatment, respectively. The PCR products of unspliced and spliced forms had an electrophoretic mobility compatible with their predicted length of 137 and 111 bp, respectively (Fig. 3). The amplicons were excised from the gel, purified and sequenced. The obtained sequences matched with human Xbp-1 sequence present in GenBank, confirming the specificity of the assay (not shown). In MDA-MB-231 cells, tunycamicin induced early (4 h) Xbp-1 splicing while this response was delayed (8 h) in CTet-treated cells. The MCF-7 cells appeared less susceptible to Xbp-1 splicing; in fact, the spliced/unspliced ratio was always lower compared to MDA-MB-231 cells. However, the spliced Xbp-1 form was evident for tunicamycin- and CTet-treatment after 8 h (Fig. 3A). The experiments were also repeated with cells treated for 24 h with 6 M and 12 M of CTet, to test the same conditions used for microarray experiments. The results confirmed the data obtained at 24 h; moreover, the smaller PCR fragment of 111 bp associated with IRE1-mediated splicing was shown to increase in a dose-dependent manner (Fig. 3B). 006ab0faaa