Crocin Induces Apoptosis in Primary Cancer Epithelial Cells Isolated from Human Breast Tumors via Different Mechanisms in HER2- Negative or Positive Cells: A Preliminary Study

Crocin Induces Apoptosis in Primary Cancer Epithelial Cells Isolated from Human Breast Tumors via Different Mechanisms in HER2- Negative or Positive Cells: A Preliminary Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Crocin Induces Apoptosis in Primary Cancer Epithelial Cells Isolated from Human Breast Tumors via Different Mechanisms in HER2- Negative or Positive Cells: A Preliminary Study S. Zahra Bathaie, Nassim Faridi, Hamid Hydrazideh, S. Ali Hashemi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4711052/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose The anticancer effect of Crocin , a natural C20 carotenoid, has been previously demonstrated in different cancer cell lines and animal cancer models. Herein, we investigated its effect on primary breast cancer cells isolated from women’s breast tumor samples. Methods We previously isolated and characterized epithelial breast cancer and normal cells from female patients. In this study, we treated five cancer cells and five normal cells from the same sample with Crocin. Then, the type and mechanisms of Crocin -induced cell death were studied using different techniques. Results All of these tumors were estrogen and progesterone receptor-positive. Two samples were in grade II and HER2-negative, while three others were grade III and HER2-positive. The IC50 of Crocin were obtained using MTT assay for all cells. It induced procaspase-9 expression and cleavage, sub-G1 accumulation, XBP1 mRNA splicing and expression of the spliced XBP1, LC3-II accumulation, and accumulation of unprenylated Rap1α in all cancer cells. The p27 mRNA expression was only induced in cells isolated from HER2-negative samples. However, an increase in the p27 protein level was observed in all cells. Crocin also down-regulated the CXCR-4 and suppressed EpCAM in these cancer cells. The unfarnesylated Lamin B was observed only in one sample. Conclusion Crocin suppressed the proliferation of human primary epithelial breast cancer cells, enhanced stress responses, and decreased metastatic markers. There was a difference between p27 expression in HER2-negative and positive tumors. Breast Cancer Apoptosis Stress Response LC3-II Accumulation Metastatic Markers EpCAM Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Breast cancer is the first- or second-leading cause of female cancer in 95% of countries [ 1 ]. The incidence and number of lives lost to breast cancer are increasing. According to the WHO prediction, more than 3 million cases of breast cancer and 1 million deaths are predicted to occur each year worldwide by the year 2040 [ 1 ]. Four types of breast cancers are introduced based on the surface receptor expression level. These are estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2) receptors. Among them, triple-negative breast cancer is the most aggressive [ 2 ]. Even though early detection has improved clinical outcomes, many patients suffer from recurrence and metastasis to distant organs [ 3 ]. Therefore, novel therapeutics are highly needed in the clinic to treat breast cancer patients. Shreds of evidence suggest that natural products are helpful for cancer prevention and therapy [ 4 ]. They can regulate numerous cellular events and physiological processes [ 5 ]. Various in vivo and in vitro studies have shown that Crocin , a water-soluble carotenoid in nature, is an antioxidant with various biological and pharmacologic properties [ 6 – 8 ]. Furthermore, the anticancer properties of Crocin have been reported in various cancer cell lines and animal models of cancer [ 9 – 16 ]. Crocin is a multifunctional compound and acts via different mechanisms and signaling pathways. It induces apoptosis in various malignancies, such as lung cancer [ 17 ], liver cancer [ 18 ], gastric adenocarcinoma [ 10 ], bladder cancer [ 19 ], leukemia [ 20 , 21 ] and breast cancer [ 11 ]. It activates caspases, changes the expression levels of pro-apoptotic and anti-apoptotic proteins like Bax and Bcl-2 [ 10 ], and induces ROS production [ 11 ]. In addition, it induces autophagy-related apoptosis in some cancers, including breast cancer cells [ 22 ] and hepatocellular carcinoma [ 23 ]. Furthermore, Crocin inhibits Akt/mTOR activity in some cancer cells [ 23 ]. It could also inhibit cell cycle progression by down-regulating cyclin D1, p21, and p53 in rats [ 9 ] and disrupting the microtubule network [ 24 ]. Crocin also decreased telomerase activity [ 18 ] and reversed the epithelial-mesenchymal transition (EMT) [ 25 ] in cancer cell lines. However, according to our literature survey, there is no information about its effect on primary breast cancer cells and normal epithelial cells, which can be an introduction to its clinical application. Recently, we isolated five primary epithelial breast cancer cells from the breast tumors of Iranian women [ 26 ]. In the present study, we try to explore the effect of Crocin on the proliferation and apoptosis of these isolated primary breast cancers and compare them to adjacent normal breast cells. In addition, we screened the responses of these cells with different cell surface receptors against Crocin to compare and figure out the possible differences in the mechanism of action in different breast tumor types. Materials & Methods Materials Crocin was isolated and purified from Iranian saffron (Crocus Sativus L.) by the previously registered method in our Lab. (National Patent #54958, Oct. 27, 2008). All materials were of analytical grade and prepared by Sigma-Aldrich or Merck Co. Methods Cell culture Breast tissue biopsies were obtained from the tumor breast tissue after surgery of breast cancer patients using the approved protocol of the Ethics Committee of Tarbiat Modares University (36D-1392-02-17). Then, cancer and normal epithelial cells were isolated and cultured in the medium as described previously [ 26 ]. Viability assay and flow cytometry The MTT assay was performed in the presence or absence of Crocin , as described previously [ 10 ]. The isolated epithelial cancer cells (1×10 5 ) were plated and then treated with Crocin’s IC50 values at various intervals. Then, Crocin ’s extent of apoptosis induction on the cancer epithelial cells was evaluated by fluorescence-activated cell sorting (FACS) of cells stained with Annexin V- PI [ 7 ]. Flow cytometry analysis was also performed on all Crocin -treated and untreated primary breast cancer cells to determine the EpCAM expression at the cell surface. The relative percentages of cells in the G1, S, or G2/M phases of cell cycle were analyzed by flow cytometry and calculated using FlowJo [ 10 ]. RT-PCR Total pure RNA was isolated from the Crocin -treated and untreated cancer epithelial cells using a RiboExTM kit (GeneAll®, Korea). The corresponding gene-specific mRNA primer pair sets are shown in Table 1 . Table 1 The name, DNA sequence, length and melting temperature (Tm) of all the designed and used forward (F) and reverse (R) primers in this study. Primer Name Length (bp) Primer Sequence Tm (°C) HPRT 125 F 5’-CCTGGCGTCGTGATTAGTG-3’ R 5’-TCAGTCCTGTCCATAATTAGTCC-3’ 58.3 57.1 Caspase-9 168 F 5’-CCACACCCAGTGACATCTTT-3’ R 5’-AAACAGCATTAGCGACCC-3’ 53.9 58.4 p27 222 F 5’-CAAACGTAAACAGCTCGAAT-3’ R 5’-CATAATGCTACATCCAACGCT-3’ 57.4 54.3 XBP1 uXBP1:283 sXBP1:257 F 5’-TTACGAGAGAAAACTCATGGCC-3’ R 5’-GGGTCCAAGTTGTCCAGAATGC-3’ 58.4 61.72 Western blotting Western blotting using the method described previously [ 7 , 11 ] investigated the expression of procaspase-9 (pCas9), cleaved caspase-9 (cCas9), p27, CXCR4, XBP1s, LC3II, Rap-1α, Lamin B, and β-Actin. Statistical Analysis The results of three independent experiments are reported as means ± SD, and were statistically evaluated using repeated measures and ANOVA followed by Tukey’s Post Hoc analysis between different groups and at different intervals, using SPSS version 16.0. Comparisons were considered statistically significant at a 95% confidence interval, P < 0.05. Results The effect of Crocin on the viability of normal and cancer primary epithelial cells The cytotoxicity and anti-proliferative effect of varying Crocin concentrations in primary normal (N) and cancer (C) epithelial cells were studied using MTT assay at different intervals (12, 24, 48 and 72 h). Figure 1 , C1 to C5, and Table 3 indicate a variation in Crocin’s half inhibitory concentrations (IC50) in five isolated breast cancer cells at different times. It was between 2.56 and 4.6 mM after 24 h. At the same condition, the IC50 of Crocin in these normal epithelial cells was between 5.7 and 7.2 mM after 24 h (Fig. 1 , N1 to N5 and Table 3 ), about two times more than those observed in the related cancer cells. The IC50 value of Crocin in each cancer epithelial cell was used for further experiments. Table 2 The pathoclinical details of isolated breast cancer cells. Clinical parameter Isolated Cells Age ER PR HER2 Tumor Grade Lymph node status C1 40 + + - II - C2 43 + + - II + C3 40 + + + III + C4 51 + + + III + C5 58 + + + III + Table 3 IC50 values of Crocin for cancer and normal epithelial cells isolated from breast tumor tissues, after 24 h treatment. Individual Crocin IC50 (mM) Cancerous Normal C1 2.56 5.63 C2 3.58 6.65 C3 4.61 7.17 C4 4.09 6.14 C5 4.64 6.65 Crocin promotes apoptosis and induces stress in primary cancer cells The primary cancer cells were treated with their IC50 value of Crocin for up to 24 h and then were analyzed by flow cytometry. The results showed increased Annexin V positive cell populations (both early and late apoptotic cells) from 6 to 24 h. Because of the similarity in these data in all cancer cells, the data of only one isolated cancer epithelial cell is shown in Figs. 2 A to 2 D at different time intervals. Figure 2 E shows the histogram of the necrotic, early, and late apoptotic cell percentages. Although the population of both early and late apoptotic cells was significantly higher after 24 h, there were significantly ( p = 0.000) higher levels of late apoptotic cells than other populations after 24 h of Crocin treatment compared to control cells. A similar pattern was observed for all other primary cancer cells. Then, we evaluated the caspase-9 (Cas9) level, at mRNA and protein levels, as a marker of the intrinsic/ mitochondrial pathway of apoptosis. Figures 3 A and 3 B show Crocin increased CAS9 mRNA expression up to 24 h ( p ≤ 0.001) compared to the control, but most changes were observed at 6 h ( p = 0.001) in all five cancer cells. Figure 3 C indicates the Crocin -induced expression of procaspase-9 (pCas9) and activation (cleaved caspase9/ cCas9) in a time-dependent manner. Figure 3 D indicates the significant induction of pCas9 expression ( p ≤ 0.01) after 6 h of Crocin treatment compared with the controls in all five cancer cells. Figure 3 E shows a significant increase ( p = 0.000) in the cCas9, indicating increased Cas9 activity in the Crocin -treated cells compared with the control. Although significant ( p = 0.001) activation of Cas9 was observed after 6 h of Crocin treatment in C1 and C2 (Group 1 cells), it was significantly ( p = 0.000) increased in C3, C4, and C5 (Group 2) after 12 h of Crocin treatment (Fig. 3 E). Figure 3 F, cCas9/ pCas9 ratio, clearly indicates the reverse trend of changes in this parameter by time in Group 1 versus Group 2 cells. The results show more increase ( p = 0.000) at 6 h, then a decrease in Group 1, and a time-dependent increase up to 24 h in Group 2 ( p = 0.000) compared to the control. The XBP1 splicing was studied at different time intervals. Figure 4 A indicates the Crocin -induced splicing of XBP1 at the mRNA level by time. Figure 4 B shows the significant ( p = 0.000) changes in the ratio of spliced XBP1 ( XBP1s ) to the unspliced XBP1 ( XBP1u ) mRNA. Similar changes were also observed in the protein level. Figures 4 C shows the Western blot data of some proteins, including the spliced form of XBP1 (XBP1s). Figure 4 D indicates a significant ( p = 0.000) increase in the XBP1s by time, especially after 12 to 24 h. Figure 4 C indicates the overall accumulation of LC3-II in cancer cells. Figure 4 E indicates a significant ( p = 0.000) increase in the LC3-II/ LC3-I ratio after 24 h of Crocin treatment compared to untreated control cells in all five isolated cancer cells. Like the results of Cas9 activation, increasing the LC3-II/ LC3-I ratio was seen in C1 and C2 (Group 1) cancer cells after 6 and 12 h of Crocin treatment, respectively. In comparison, it was increased up to 24 h in three other cells (Group 2). Figure 4 C also shows the Western blot pattern of Lamin B expression and farnesylation in the mentioned cells. The bands related to unfarnesylated Lamin B were only seen in C1 cells. Figure 4 F shows that Lamin B expression in C1 decreased at 6 h and then increased up to 24 h. In addition, the unfarnesylated Lamin B was increased ( p = 0.000) up to 24 h of Crocin treatment. The ratios of unfarnesylated (uF)/ farnesylated (F) Lamin B are shown in the inset of Fig. 4 F. Except C1, a relatively similar pattern was observed in the Lamin B expression in four other breast cancer cells (C2 to C5), which their histogram is shown in Fig. 4 G. Although the level of Lamin B was different before Crocin treatment (time 0), it was leveled off by time ( p = 0.000) up to 24 h in these cells. Regulation of the cell progression and metastasis prevention by Crocin To elucidate whether cell cycle arrest is also involved in the growth inhibitory effect of Crocin , we evaluated the cell cycle distribution by flow cytometry (Fig. 5 ). As shown in Fig. 5 A and 5 B indicate that the population of cells at the S phase significantly ( p = 0.000) decreased after 12 h of Crocin treatment, in all cancer cells except C1. After 24 h of Crocin treatment, the percentages of sub-G1 (apoptotic) cells significantly increased ( p = 0.000) in all isolated cancer cells. So, over 50% of the C1 and C2 cell populations were in the sub-G1 phase after 24 h. These changes were accompanied by a significant ( p = 0.005) reduction of all cell accumulation in the G0/G1 phase after 24 h. These results suggest that Crocin induces G0/G1- and S-phase cell cycle arrest in the isolated breast cancer cells. Consequently, we evaluated the effect of Crocin on the expression of p27, a cyclin-dependent kinase inhibitor involved in the cell cycle arrest at the G1 phase (Figs. 6 A and 6 D). Although the expression of p27 mRNA was increased significantly in C1 and C2 cancer cells, after 6 h of Crocin treatment ( p = 0.000), there were no significant changes in the expression of p27 mRNA in Group 2 (C3, C4, and C5) cells. The results of p27 protein expression are shown in Figs. 6 C and 6 D. In contrast to the unchanged p27 mRNA levels in these cells, the level of p27 protein significantly increased ( p ≤ 0.001) in Group 2 cancer cells up to 24 h of Crocin treatment. On the other hand, a significant increase in the p27 mRNA expression at 6 h in C1 and C2 cancer cells did not translate to such a large protein production. We also examined the expression of the CXCR4 protein involved in cancer progression and metastasis. Figures 6 C and 6 E indicate significant ( p = 0.000) and time-dependent decrease, in the CXCR4 levels, especially after 24 h of Crocin treatment. Figure 6 C shows the effect of Crocin on the accumulation of unprenylated (uP)-Rap-1α up to 24 h after treatment. Figure 6 F shows the histograms of Rap-1α expression in these cells. It indicates a similar pattern in all five cells and increased accumulation of unprenylated uP-Rap-1α in the Crocin -treated cells compared to the control. However, the unprenylated form of this protein was significantly higher (p < 0.01) in Group 2 cells up to 24 h than in Group 1 cells (p < 0.05). We also monitored the effect of Crocin on EpCAM expression, another marker for cell growth and metastasis. Figures 7 A and 7 B showed that Crocin treatment decreased the Ep-CAM expression after 24 h in all Crocin -treated breast cancer cells compared to the control. Discussion We investigated the mechanism(s) of the anticancer effect of Crocin in the primary breast cancer cells isolated from Iranian women [ 26 ]. According to our literature survey, it is the first report related to the anticancer activity of Crocin on primary breast cancer cells. Thus, the effect of Crocin on both HER2/neu negative cells, Group 1 (C1 and C2), and HER2/neu positive cells, Group 2 (C3, C4, and C5), was investigated. Crocin significantly inhibited the growth of these cancer epithelial cells and induced death with different IC50, possibly based on tumor grade, surface receptors, and other intrinsic/ genetic features of each cell. The mechanism of cell death induction was apoptosis through Cas9 upregulation and activation in all five isolated breast cancer cells. Crocin also induced some unfolded protein response (UPR) and autophagy markers In addition, it decreased cancer cell proliferation by retaining the primary cancer cells in the sub-G1 phase and preventing them from entering the G0/G1 phase, especially in Group 1 cells that were HER2/neu negative. An overall increase in the p27 protein level was observed up to 24 h of Crocin treatment. Especially in HER2/neu positive cells. Crocin also decreased the expression of CXCR4 and EpCAM in these cancer cells. As mentioned in the introduction, we previously isolated and characterized cancer and normal epithelial cells from the breast cancer tumors of five patients [ 26 ]. We treated these cells with Crocin in the present study and determined their IC50 using an MTT assay. The tumors used in this study were categorized according to breast cancer grade and the expression of surface hormone receptors on the cells (Table 2 ). Group 1 (C1 and C2), were grade II breast cancer cases and HER2/neu negative. Group 2 (C3, C4, and C5), were grade III cases and HER2/neu positive. The lymph nodes were involved in all cases except C1. The grade II breast cancer cells were more sensitive to Crocin than grade III cases. Cell death was induced with lower doses of this natural carotenoid in these cells than in grade II cells. We previously determined the IC50 of Crocin in various breast cancer cell lines. It has obtained 2.7 mM in MDA-MB-231 [ 11 ], 3.0 mM in MCF-7 [ 11 ], 3.1 in MDA-MB-468 [ 22 ], and 3.6 mM in BT-474 [ 27 ]. As mentioned in our previous review paper, the IC50 of Crocin has been reported up to 5.5 mM in various cancer cells [ 28 ]. On the other hand, our previous in vivo studies indicated that four-weeklies i.p. injection of 150 mg/Kg body weight Crocin efficiently removed tumors or reduced tumor sizes in the NMU-induced breast cancer in rats [ 9 ] and in the 4T1-induced breast cancer in BALB/c mice [ 29 ]. The in vitro colony-formation assay also indicated the effectiveness of Crocin at µM concentration in breast cancer cells [ 11 ]. Thus, in contrast to the high Crocin concentrations needed to induce apoptosis in various cancer cells, the in vivo dose needed to suppress the tumor growth is significantly low. Although the safety of Crocin has been reported for human [ 30 , 31 ] and in normal animals [ 32 ], its effect on normal epithelial breast cell has not been reported. In some studies, MCF-10 [ 33 ], which is not a normal breast cell, and in some others, fibroblast cells [ 10 ] with entirely different genetic patterns from the breast epithelial cells have been used. Thus, in the present study, we applied the primary breast normal cells isolated from the adjacent tissues of tumors to compare the results. The data indicated no toxicity of Crocin for normal breast cells of the same tissue at the toxic dosages obtained for those cancer cells. Cancer cells were significantly more susceptible to Crocin treatment than normal cells. Continuing the mechanistic study, we reported a Crocin -induced Cas9 expression in five isolated breast cancer cells. Additionally, Crocin induced the cleavage of pCas9 and the production of cCas9 in these cancer cells. These results agree with previous studies showing the apoptotic effect of Crocin in various breast cancer cell lines [ 11 , 27 , 34 – 36 ] and the role of mitochondria-mediated apoptosis, which is precisely regulated by the Bcl-2 family proteins [ 10 , 11 ]. Furthermore, the inhibitory role of Crocin on the growth of other human cancer cells [ 10 , 17 , 20 , 37 ] and in animal models of breast and gastric cancers [ 9 , 38 , 39 ] could be attributed to a similar mechanism. Another method to approve apoptotic cell death is to detect and quantify the percentages of apoptotic or necrotic cells by annexin V/ PI staining using flow cytometry [ 40 ]. Our results showed a similar apoptotic pattern in all Crocin -treated isolated breast cancer cells, with significantly higher populations of apoptotic cells than necrotic cells. Activation of the UPR as a result of the endoplasmic reticulum (ER) stress has a crucial role in protein homeostasis and other diverse functions involved in the process of breast cancer progression. In this regard, we studied the impact of Crocin on XBP1 slicing , one of the ER stress indicators [ 41 ]. After Crocin treatment, the XBP1 mRNA splicing increased by time, which ultimately resulted in the expression of the spliced protein (XBP1s) in all five breast cancer cells. Similar changes has been reported in MDA-MB-468 and BT-474 breast cancer cell lines after Crocin treatment [ 42 ]. Here, we observed the inductive effect of Crocin on LC3II production and accumulation, which was accompanied by an increase in the LC3II/LC3I ratio in all five cancer cells. We have previously shown that Crocin changed the LC3II/LC3I ratio in MDA-MB-468 and MCF-7 breast cancer cell lines [ 22 , 42 ]. The conversion of LC3I to LC3II has been introduced as an autophagy marker [ 43 ]. Our data also showed a time-dependent decrease of Lamin B protein in these primary breast cancer cells after Crocin treatment. It has been shown that LC3 directly interacts with Lamin B1, and then this complex bind to Lamin-associated chromatin domains. These interactions cause the autophagy-mediated destruction of the nuclear lamina. The nuclear lamina degradation impairs cell proliferation by inducing cell-cycle arrest as a tumor-suppressive mechanism [ 44 ]. UPR and autophagy have been known as adaptive mechanisms to regulate cellular function during stress. If the stress is prolonged, apoptotic cell death ensues [ 41 , 45 , 46 ]. Recently, we showed a Crocin -induced ROS production in cancer cells [ 11 ]. In addition, the process can induce UPR-regulated autophagy and apoptosis in tumor cells [ 27 ]. A similar phenomenon, autophagic-induced apoptosis, has been reported as the mechanism of action of other anticancer compounds in other types of cancer [ 47 – 49 ]. As an antitumor agent, Crocin induced cell cycle arrest via changing p53, p21, and cyclin D1 in the NMU-induced breast cancer in rats [ 9 ]. The data in the present study also indicates the cell-cycle arrest induction at the G0/G1 phase after Crocin treatment in primary breast cancer cells. A similar role of Crocin has also been reported in the human gastric cancer cell line [ 38 ]. Crocin retains cells in the sub-G1 phase and decreased their entry into S phases, especially in HER2/neu positive cells. To investigate the effect of Crocin on cell cycle regulators, we examined the expression of p27. Similar to the present study, a reverse relation between p27 and HER2/neu expression has been shown in breast cancer [ 50 ]. HER2/neu signals caused a decrease in p27 stability and enhancement of its degradation. By blocking HER2/neu, p27 has been upregulated [ 51 ]. So, in C1 and C2 cancer cells (grade II and HER2/neu negative), the p27 mRNA expression was higher than in Group 2 cancer cells (grade III and HER2/neu positive). Although Crocin treatment in HER2/neu positive cells did not significantly alter p27 mRNA levels, the protein levels of p27 increased in these cancer cells. This effect may be due to Crocin ’s ability to overcome the HER2/neu or its downstream signaling pathway and inhibition of p27 degradation. All primary breast cancer cells in this study were isolated from patients with ductal carcinoma in situ. As the data show, EpCAM was expressed in all of them. EpCAM has been known to be overexpressed in epithelial cancer cells, and its overexpression appears to be associated with enhanced proliferation and malignant potential [ 52 , 53 ]. Crocin inhibited the EpCAM expression and was more effective in grade II tumor cells than in grade III. It is conceivable that HER2/neu-independent mechanisms may be responsible for the downregulation of EpCAM in Crocin -treated breast cancer cells. A link between uP-Rap-1α and cell proliferation and tumor cell migration and invasion has been extensively studied and reviewed [ 54 ]. Furthermore, the diminished Rap-1α expression decreased cell migration ability [ 55 ]. Our data also indicated the increased uP-Rap-1α due to the Crocin treatment of all breast cancer cells. However, the changes were different in Group 1 and Group 2 cancer cells. Before Crocin treatment, the accumulation of uP-Rap-1α in Group 2 cancer cells was significantly lower than in Group 1. Crocin induced the accumulation of uP-Rap-1α more than 4-fold in Group 2 cancer cells compared with Group 1, which was increased less than 1-fold. So, after 12 h of Crocin treatment, all five cancer cells reached the same level of uP-Rap-1 accumulation. A significant correlation between HER2 and CXCR4 expression has been observed in human breast tumor tissues, related to cancer recurrence, metastasis, and poor survival rates [ 56 ]. Furthermore, the degree of CXCR4 expression, a chemokine signaling system important in breast cancer progression and metastasis [ 57 , 58 ], was also higher in HER2/neu positive (Group 2) than HER2/neu negative (Group 1) cancer cells. Here, we observed that Crocin significantly decreased the expression of CXCR4 in all five breast cancer cells. However, it was more effective in the HER2/neu positive group than the HER2/neu negative cells. It indicates that Crocin might inhibit the HER2/neu signaling and its association with metastasis in breast cancer cells. The limitations of this study include the limited number of primary breast cells. Thus, it should continue to use a larger population of primary breast cancer cells with different genetic characteristics. Furthermore, a clinical trial should be designed for Crocin application as a supplement in breast cancer patients. However, it shows that tumor genetics is essential in the Crocin ’s anticancer mechanism(s). Therefore, this subject is crucial in precision medicine and should be considered in future studies. Conclusion In the present study, XBP1 splicing and LC3-II accumulation, MTT assay, flow cytometry data, and the pCas9 overexpression and activation confirmed that Crocin could induce stress in cancer cells. When the stress was prolonged, apoptotic cell death occurred in a time-dependent manner in all five breast cancer cells. LC3-II accumulation, Lamin B expression, and prenylation were also investigated as the factors involved in the autophagy-mediated destruction of the nuclear lamina. This study highlighted the importance of breast cancer cell characteristics and response to treatment. Since the cells were genetically different, different doses of Crocin induced death in the isolated cancer cells through two different mechanisms of apoptosis and cell cycle arrest. It also decreased the expression of proteins involved in proliferation and metastasis in breast cancer cells isolated from biopsies with HER2/neu positive rather than HER2/neu negative. The effect of Crocin depends explicitly on the proteins of HER2/neu and its downstream signaling involved in proliferation and metastasis. Thus, by studying each patient’s biopsy and investigating the genetic/ protein patterns, we can determine the amount of Crocin needed to induce stress and, finally, apoptosis and activate different mechanisms for cell death induction in tumor cells. Declarations Funding Elite Researcher Grant Committee of the National Institute for Medical Research Development (NIMAD) supported the research reported in this publication under award numbers [971400], Tehran, Iran. Author Contribution S.Z.B.: Supervised the study; Conception and design of the work; Analysis; Interpretation of data, Editing and Revising the manuscript.N.F.: Conception and Design; Data acquisition, Analysis; Interpretation of data; Drafting the manuscript.H.H.: Study Design, Western blotting, and Data Analysis.S.A.H.: Study Design, Data acquisition and Analysis.S.A.: Conception and Design, Data interpretation, Editing the manuscript.F.T.: Conception and Design, Data interpretation, Editing the manuscript.M.-A.M.: Conception and Design, Clinical Control; manuscript evaluation.All authors reviewed the manuscript. Acknowledgement The authors thank the “Iran Science Elites Federation” for supporting Professor Bathaie and granting Dr. Faridi a Postdoc. Data Availability The raw data for Western blot analysis will be sent for review if requested. 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Asian Pac J cancer prevention: APJCP 13(5):2305–2309 Zhao P, Luo CL, Wu XH, Hu HB, Lv CF, Ji HY (2008) [Proliferation apoptotic influence of crocin on human bladder cancer T24 cell line]. Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China J Chin materia Med 33(15):1869–1873 Sun Y, Wang Z, Wang L, Wang LZ, Zang C, Sun LR (2015) The Effect and Mechanisms of Proliferative Inhibition of Crocin on Human Leukaemia Jurkat Cells. West Indian Med J 64(5):473–479 Rezaee R, Mahmoudi M, Abnous K, Zamani Taghizadeh Rabe S, Tabasi N, Hashemzaei M, Karimi G (2013) Cytotoxic effects of crocin on MOLT-4 human leukemia cells. J Complement Integr Med 10 Heidarzadeh H, Bathaie SZ, Abroun S, Mohagheghi MA (2018) Evaluating the cytotoxic effect of crocin on MDA-MB-468 cell line based on apoptosis induction, ER stress, and autophagy markers. Pathobiology Res 20(4):37–51 Yao C, Liu BB, Qian XD, Li LQ, Cao HB, Guo QS, Zhou GF (2018) Crocin induces autophagic apoptosis in hepatocellular carcinoma by inhibiting Akt/mTOR activity. OncoTargets therapy 11:2017–2028 Hire RR, Srivastava S, Davis MB, Kumar Konreddy A, Panda D (2017) Antiproliferative Activity of Crocin Involves Targeting of Microtubules in Breast Cancer Cells. Sci Rep 7:44984 Festuccia C, Mancini A, Gravina GL, Scarsella L, Llorens S, Alonso GL, Tatone C, Di Cesare E, Jannini EA, Lenzi A et al (2014) Antitumor effects of saffron-derived carotenoids in prostate cancer cell models. Biomed Res Int 2014:135048 Faridi N, Bathaie SZ, Abroun S, Farzaneh P, Karbasian H, Tamanoi F, Mohagheghi MA (2018) Isolation and characterization of the primary epithelial breast cancer cells and the adjacent normal epithelial cells from Iranian women's breast cancer tumors. Cytotechnology 70(2):625–639 Faridi N, Heidarzadeh H, Mohagheghi MA, Bathaie SZ (2019) BT-474 breast cancer cell apoptosis induced by crocin, a saffron carotenoid. Basic Clin Cancer Res 11(1):5–15 Bolhassani A, Khavari A, Bathaie SZ (2014) Saffron and natural carotenoids: Biochemical activities and anti-tumor effects. Biochim Biophys Acta 1845(1):20–30 Salarifar A (2019) Investigation of Crocin effect on induced breast cancer tumors in Balb/c mice: expression of caspase-9 and CXCR4. Tarbiat Modares University, Iran Mousavi B, Bathaie SZ, Fadai F, Ashtari Z, Ali Beigi N, Farhang S, Hashempour S, Shahhamzei N, Heidarzadeh H (2015) Safety evaluation of saffron stigma (Crocus sativus L.) aqueous extract and crocin in patients with schizophrenia. Avicenna J phytomedicine 5(5):413–419 Mohamadpour AH, Ayati Z, Parizadeh MR, Rajbai O, Hosseinzadeh H (2013) Safety evaluation of crocin (a constituent of saffron) tablets in healthy volunteers. Iran J basic Med Sci 16(1):39 Taheri F, Bathaie SZ, Ashrafi M, Ghasemi E (2014) Assessment of crocin toxicity on the rat liver. Pathobiology Res 17(3):67–79 Qu Y, Han B, Yu Y, Yao W, Bose S, Karlan BY, Giuliano AE, Cui X (2015) Evaluation of MCF10A as a reliable model for normal human mammary epithelial cells. PLoS ONE 10(7):e0131285 Hashemi SA, Bathaie SZ, Mohagheghi MA (2019) Interaction of saffron carotenoids with catalase: in vitro, in vivo and molecular docking studies. J Biomol Struct Dyn :1–11 Jia Y, Yang H, Yu J, Li Z, Jia G, Ding B (2024) Crocin enhances the sensitivity to paclitaxel in human breast cancer cells by reducing BIRC5 expression. Chem Biol Drug Des 103(2):e14467 Zhu J, Zheng S, Liu H, Wang Y, Jiao Z, Nie Y, Wang H, Liu T, Song K (2021) Evaluation of anti-tumor effects of crocin on a novel 3D tissue-engineered tumor model based on sodium alginate/gelatin microbead. Int J Biol Macromol 174:339–351 Kim B, Lee KY, Park B (2017) Crocin Suppresses Constitutively Active STAT3 Through Induction of Protein Tyrosine Phosphatase SHP-1. J Cell Biochem 118(10):3290–3298 Bathaie SZ, Miri H, Mohagheghi MA, Mokhtari-Dizaji M, Shahbazfar AA, Hasanzadeh H (2013) Saffron Aqueous Extract Inhibits the Chemically-induced Gastric Cancer Progression in the Wistar Albino Rat. Iran J Basic Med Sci 16(1):27–38 Sajjadi M, Bathaie Z (2017) Comparative Study on The Preventive Effect of Saffron Carotenoids, Crocin and Crocetin, in NMU-Induced Breast Cancer in Rats. Cell J 19(1):94–101 Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 184(1):39–51 Szegezdi E, Logue SE, Gorman AM, Samali A (2006) Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep 7(9):880–885 Heidarzadeh H (2017) Effect of Crocin and Crocetin Obtained from Saffron (Crocus Sativus L.) on ER-Stress and Autophagy markers (XBP1s and LC3-II, respectively) on the MCF7 and MDA-MB-468 Breast Cancer Cells. Tarbiat Modares University Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19(21):5720–5728 Dou Z, Xu C, Donahue G, Shimi T, Pan JA, Zhu J, Ivanov A, Capell BC, Drake AM, Shah PP et al (2015) Autophagy mediates degradation of nuclear lamina. Nature 527(7576):105–109 Kondo Y, Kanzawa T, Sawaya R, Kondo S (2005) The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 5(9):726–734 Ogata M, Hino S, Saito A, Morikawa K, Kondo S, Kanemoto S, Murakami T, Taniguchi M, Tanii I, Yoshinaga K et al (2006) Autophagy is activated for cell survival after endoplasmic reticulum stress. Mol Cell Biol 26(24):9220–9231 Bhutia SK, Dash R, Das SK, Azab B, Su ZZ, Lee SG, Grant S, Yacoub A, Dent P, Curiel DT et al (2010) Mechanism of autophagy to apoptosis switch triggered in prostate cancer cells by antitumor cytokine melanoma differentiation-associated gene 7/interleukin-24. Cancer Res 70(9):3667–3676 Rovetta F, Stacchiotti A, Consiglio A, Cadei M, Grigolato PG, Lavazza A, Rezzani R, Aleo MF (2012) ER signaling regulation drives the switch between autophagy and apoptosis in NRK-52E cells exposed to cisplatin. Exp Cell Res 318(3):238–250 Trincheri NF, Follo C, Nicotra G, Peracchio C, Castino R, Isidoro C (2008) Resveratrol-induced apoptosis depends on the lipid kinase activity of Vps34 and on the formation of autophagolysosomes. Carcinogenesis 29(2):381–389 Filipits M, Dafni U, Gnant M, Polydoropoulou V, Hills M, Kiermaier A, de Azambuja E, Larsimont D, Rojo F, Viale G et al (2018) Association of p27 and Cyclin D1 Expression and Benefit from Adjuvant Trastuzumab Treatment in HER2-Positive Early Breast Cancer: A TransHERA Study. Clin cancer research: official J Am Association Cancer Res 24(13):3079–3086 Yang HY, Zhou BP, Hung MC, Lee MH (2000) Oncogenic signals of HER-2/neu in regulating the stability of the cyclin-dependent kinase inhibitor p27. J Biol Chem 275(32):24735–24739 de Boer CJ, van Krieken JH, Janssen-van Rhijn CM, Litvinov SV (1999) Expression of Ep-CAM in normal, regenerating, metaplastic, and neoplastic liver. J Pathol 188(2):201–206 Wang R, Yang L, Li S, Ye D, Yang L, Liu Q, Zhao Z, Cai Q, Tan J, Li X (2018) Quercetin Inhibits Breast Cancer Stem Cells via Downregulation of Aldehyde Dehydrogenase 1A1 (ALDH1A1), Chemokine Receptor Type 4 (CXCR4), Mucin 1 (MUC1), and Epithelial Cell Adhesion Molecule (EpCAM). Med Sci Monit 24:412–420 Zhang YL, Wang RC, Cheng K, Ring BZ, Su L (2017) Roles of Rap1 signaling in tumor cell migration and invasion. Cancer biology Med 14(1):90–99 Zhang T, Jiang K, Zhu X, Zhao G, Wu H, Deng G, Qiu C (2018) miR-433 inhibits breast cancer cell growth via the MAPK signaling pathway by targeting Rap1a. Int J Biol Sci 14(6):622 Li YM, Pan Y, Wei Y, Cheng X, Zhou BP, Tan M, Zhou X, Xia W, Hortobagyi GN, Yu D et al (2004) Upregulation of CXCR4 is essential for HER2-mediated tumor metastasis. Cancer Cell 6(5):459–469 Kang H, Watkins G, Parr C, Douglas-Jones A, Mansel RE, Jiang WG (2005) Stromal cell derived factor-1: its influence on invasiveness and migration of breast cancer cells in vitro, and its association with prognosis and survival in human breast cancer. Breast cancer research: BCR 7(4):R402–410 Luker KE, Luker GD (2006) Functions of CXCL12 and CXCR4 in breast cancer. Cancer Lett 238(1):30–41 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4711052","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":335589990,"identity":"022ef9f6-e187-425f-adce-f07507088828","order_by":0,"name":"S. Zahra Bathaie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIie2RPwrCMBSHnwjpEsiaUg8RESJFMVexBDp18AidnHoAwas4PCm4uoniYhE69QAKBY1/UBdT3BzyDeEl8PF7PwLgcPwhAoDcT2Yu/PU8tirth+Knvym3AT8VG31vvTqcJqB6m3y5PS+GCrz8AMXiuxJm2utmJkvuYx1mZRylNBYwLi2LoSacCiByn0hOMTctEtMFLcr6SPxaAO3NE+nXeFHAqgZlq0lgUrgIEhlQxFbKG1LC2VEGHQGCmy6DDupoykuBNqXPotKvalBsrpe7CkeKMV0UJ4vy5PIeb9/ULDgcDofDyhXoIEkkwfnMvgAAAABJRU5ErkJggg==","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":true,"prefix":"","firstName":"S.","middleName":"Zahra","lastName":"Bathaie","suffix":""},{"id":335589993,"identity":"2eae6624-fdca-49d9-90b6-81587bef8c31","order_by":1,"name":"Nassim Faridi","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"Nassim","middleName":"","lastName":"Faridi","suffix":""},{"id":335589994,"identity":"2bffa4fa-77c6-42bd-a305-65f8a46fc9ee","order_by":2,"name":"Hamid Hydrazideh","email":"","orcid":"","institution":"Institute for Natural Products and Medicinal Plants, Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"Hamid","middleName":"","lastName":"Hydrazideh","suffix":""},{"id":335589995,"identity":"63aae821-ee6d-47f9-8221-e84994afe95e","order_by":3,"name":"S. Ali Hashemi","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"S.","middleName":"Ali","lastName":"Hashemi","suffix":""},{"id":335589996,"identity":"82f33cd9-4fb2-4c57-882a-ba5d672a858c","order_by":4,"name":"Saeid Abroun","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"Saeid","middleName":"","lastName":"Abroun","suffix":""},{"id":335589998,"identity":"64d8adf7-43e4-4857-a1be-f900048db294","order_by":5,"name":"Fuyuhiko Tamanoi","email":"","orcid":"","institution":"University of California, Los Angeles, UCLA","correspondingAuthor":false,"prefix":"","firstName":"Fuyuhiko","middleName":"","lastName":"Tamanoi","suffix":""},{"id":335589999,"identity":"de54695f-61e8-4cd8-b84c-327488e31c6f","order_by":6,"name":"Mohammad-Ali Mohagheghi","email":"","orcid":"","institution":"Imam Khomeini Hospital, Tehran University of Medical Science","correspondingAuthor":false,"prefix":"","firstName":"Mohammad-Ali","middleName":"","lastName":"Mohagheghi","suffix":""}],"badges":[],"createdAt":"2024-07-09 09:57:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4711052/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4711052/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62189014,"identity":"eebb163b-5b8e-4cad-ac3c-5f9128ec7325","added_by":"auto","created_at":"2024-08-10 12:19:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":481170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCrocin\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e on cell viability of different epithelial cells as determined by MTT assay.\u003c/strong\u003e The IC\u003csub\u003e50\u003c/sub\u003e of \u003cem\u003eCrocin\u003c/em\u003e for each cell was obtained using the plots. The isolated primary cancer (C1 to C5) and normal (N1 to N5) epithelial cells were incubated with increasing concentrations (1– 5 mM and 1-10 mM for cancer and normal cells, respectively) of \u003cem\u003eCrocin\u003c/em\u003e at different time intervals of 0, 12, 24, 48 and 72 h. The exact number of C or N indicates the same patient. Each value is presented as mean ± SD of three independent repeats of experiments.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4711052/v1/105227e46927f4a095cc4c51.png"},{"id":62189013,"identity":"6a943437-16a1-42bd-9c16-440021df0a45","added_by":"auto","created_at":"2024-08-10 12:19:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":460920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe flow cytometry data obtained using annexin V-FITC and PI staining.\u003c/strong\u003e (A) Shows the result of C1 primary cells without treatment (control). (B to D) show the results of C1 primary cells after 6, 12, and 24 h treatment with \u003cem\u003eCrocin\u003c/em\u003e, respectively. In each panel, the lower left quadrant shows the percentages of alive cells negative for both PI and annexin V-FITC; the upper left quadrant shows only PI-positive cells, necrotic; the lower right quadrant shows annexin-positive cells, early apoptotic; and the upper right quadrant shows annexin and PI positive cells, late apoptotic cells. (E) The histogram represents the percentage of necrotic, early, and late apoptotic cells of C1 cells after different hours of incubation with IC50 concentration of \u003cem\u003eCrocin\u003c/em\u003e. Significant changes were observed in both early and late apoptosis after 24 h of \u003cem\u003eCrocin\u003c/em\u003e treatment. * Represents P \u0026lt; 0.01 vs. control.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4711052/v1/6d4ecae7d6733ed936a5fb66.png"},{"id":62189017,"identity":"b5477cac-21ce-4fae-9ec2-676f8569caae","added_by":"auto","created_at":"2024-08-10 12:19:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":431304,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCrocin\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eon caspase-9 expression in cancer epithelial cells.\u003c/strong\u003e (A and B), RT-PCR analysis of \u003cem\u003eCASP9\u003c/em\u003e and \u003cem\u003eHPRT\u003c/em\u003e gene expression after treatment with \u003cem\u003eCrocin\u003c/em\u003eat 6, 12, and 24 h. (C) Western blot analysis of the pro-caspase-9 (pCas9) and β-Actin expression and activation in all epithelial cancer cells of C1 to C5. (D, E, and F) The histograms of pCas9/ β-Actin ratio, cleaved caspase-9 (cCas9)/ β-Actin ratio and cCas9/ pCas9 ratio, respectively, in these cells. All represented data are means ± S.D. of three independent experiments (*P \u0026lt; 0.05 vs. control).\u003c/p\u003e","description":"","filename":"Fig.3Ed.png","url":"https://assets-eu.researchsquare.com/files/rs-4711052/v1/02c689c1032419b243f014f9.png"},{"id":62190367,"identity":"392a0159-798b-4dcb-9731-7cc01d5b0db4","added_by":"auto","created_at":"2024-08-10 12:27:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":455848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression pattern of some proteins in the cancer cells after \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCrocin\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e treatment.\u003c/strong\u003e (A) RT-PCR analysis of XBP1 mRNA splicing after 6, 12, and 24 h of \u003cem\u003eCrocin\u003c/em\u003e treatment. (B) The histogram of the \u003cem\u003eXBP1s\u003c/em\u003e/ \u003cem\u003eXBP1u\u003c/em\u003e mRNA ratio was analyzed by using ImageJ. (C) Western blot bands of five isolated cancer cells after 6, 12, and 24 h of treatment with IC\u003csub\u003e50\u003c/sub\u003e doses of \u003cem\u003eCrocin\u003c/em\u003e using different antibodies. Cell lysates were analyzed by Western blot for XBP1, LC3, and Lamin B. β-Actin was used as a loading control. (D, E, and F) The histograms of XBP1s/ β-Actin ratio, LC3II/LC3I ratio, and Lamin B/ β-Action ratio were obtained by Image J analysis of Western blot bands. The inset of Fig. F shows the increase in the unfarnesylated Lamin B in C1 over time. (G) shows reduction in Lamin B/β-Actin ratio over time in C2, C3, C4 and C5 cells. Represented data are mean ± SD of three independent experiments (*P \u0026lt; 0.01 and **P \u0026lt; 0.05 vs. control).\u003c/p\u003e","description":"","filename":"Fig.4Ed.png","url":"https://assets-eu.researchsquare.com/files/rs-4711052/v1/317ac2543e95eb56c42a1feb.png"},{"id":62190878,"identity":"9405d3f0-0ed3-491f-b0d1-cfa7afe81340","added_by":"auto","created_at":"2024-08-10 12:35:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":395249,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell cycle profiles of the isolated cancer epithelial cells after \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCrocin\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e treatment as obtained by flow cytometry (PI staining).\u003c/strong\u003e (A) The cell cycle distribution of the named cells at different time intervals of 0, 12 and 24 h after \u003cem\u003eCrocin\u003c/em\u003e treatment. (B) The histograms of cell cycle distribution in cancer cells are represented in front of each cell. Values are means ± S.D. of three independent experiments conducted in triplicate. *p\u0026lt;0.05 when compared with control.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4711052/v1/ca3f0a815be3c865b77c3883.png"},{"id":62189020,"identity":"088ebdce-e2bf-42a5-be91-feb53dba90a2","added_by":"auto","created_at":"2024-08-10 12:19:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":427336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression patterns of p27, CXCR4 and Rap-1α in epithelial cancer cells.\u003c/strong\u003e (A) RT-PCR analysis of \u003cem\u003ep27\u003c/em\u003e in C1 to C5 cells treated with \u003cem\u003eCrocin\u003c/em\u003e at 0, 6, 12 and 24 h, \u003cem\u003eHPRT\u003c/em\u003e was used as a loading control. (B) Histogram of p27/HPRT mRNA ratios in different cells at different time intervals. (C) Western blot bands of p27, CXCR4, Rap-1α, and β-Actin at different time intervals after \u003cem\u003eCrocin\u003c/em\u003etreatment. β-Actin was used as an internal control. (D, E, and F) The histograms of expression ratios of p27/ β-Actin, CXCR4/ β-Actin, and unprenylated- (uP)-Rap-1α/ β-Actin as obtained by Image J analysis. Represented data are mean ± SD of three independent experiments (*P \u0026lt; 0.01, **P \u0026lt; 0.05 vs. control).\u003c/p\u003e","description":"","filename":"Fig.6Ed.png","url":"https://assets-eu.researchsquare.com/files/rs-4711052/v1/eaa05fc27ff9f17b37bdbd38.png"},{"id":62189015,"identity":"051c3541-2abc-4b4b-8452-5ea2ba54156a","added_by":"auto","created_at":"2024-08-10 12:19:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":121361,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Ep-CAM expression in the cells treated with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCrocin\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and the untreated cells after 24 h\u003c/strong\u003e. It was measured by PE-labeled anti-EpCAM antibody using flow cytometry. (A) Ep-CAM expression in five isolated cancer cells with or without \u003cem\u003eCrocin\u003c/em\u003e treatment.\u003cstrong\u003e \u003c/strong\u003e(B) Histograms of the percentages of Ep-CAM expression. Represented data are mean ± SD of three independent experiments (*P \u0026lt; 0.01, **P \u0026lt; 0.05 vs. control).\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-4711052/v1/c69730a0eba0bb4275df1a0a.png"},{"id":62382868,"identity":"5a91b559-fcb9-4c34-88e0-9f127461c6fd","added_by":"auto","created_at":"2024-08-13 14:31:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3783061,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4711052/v1/cca2f367-c207-4ad7-b9a6-35b4b7531d23.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Crocin Induces Apoptosis in Primary Cancer Epithelial Cells Isolated from Human Breast Tumors via Different Mechanisms in HER2- Negative or Positive Cells: A Preliminary Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer is the first- or second-leading cause of female cancer in 95% of countries [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The incidence and number of lives lost to breast cancer are increasing. According to the WHO prediction, more than 3\u0026nbsp;million cases of breast cancer and 1\u0026nbsp;million deaths are predicted to occur each year worldwide by the year 2040 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFour types of breast cancers are introduced based on the surface receptor expression level. These are estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2) receptors. Among them, triple-negative breast cancer is the most aggressive [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Even though early detection has improved clinical outcomes, many patients suffer from recurrence and metastasis to distant organs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, novel therapeutics are highly needed in the clinic to treat breast cancer patients.\u003c/p\u003e \u003cp\u003eShreds of evidence suggest that natural products are helpful for cancer prevention and therapy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. They can regulate numerous cellular events and physiological processes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Various \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e studies have shown that \u003cem\u003eCrocin\u003c/em\u003e, a water-soluble carotenoid in nature, is an antioxidant with various biological and pharmacologic properties [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, the anticancer properties of \u003cem\u003eCrocin\u003c/em\u003e have been reported in various cancer cell lines and animal models of cancer [\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13 CR14 CR15\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eCrocin\u003c/em\u003e is a multifunctional compound and acts via different mechanisms and signaling pathways. It induces apoptosis in various malignancies, such as lung cancer [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], liver cancer [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], gastric adenocarcinoma [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], bladder cancer [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], leukemia [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and breast cancer [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It activates caspases, changes the expression levels of pro-apoptotic and anti-apoptotic proteins like Bax and Bcl-2 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and induces ROS production [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, it induces autophagy-related apoptosis in some cancers, including breast cancer cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and hepatocellular carcinoma [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Furthermore, \u003cem\u003eCrocin\u003c/em\u003e inhibits Akt/mTOR activity in some cancer cells [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It could also inhibit cell cycle progression by down-regulating cyclin D1, p21, and p53 in rats [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and disrupting the microtubule network [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. \u003cem\u003eCrocin\u003c/em\u003e also decreased telomerase activity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and reversed the epithelial-mesenchymal transition (EMT) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] in cancer cell lines. However, according to our literature survey, there is no information about its effect on primary breast cancer cells and normal epithelial cells, which can be an introduction to its clinical application.\u003c/p\u003e \u003cp\u003eRecently, we isolated five primary epithelial breast cancer cells from the breast tumors of Iranian women [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the present study, we try to explore the effect of \u003cem\u003eCrocin\u003c/em\u003e on the proliferation and apoptosis of these isolated primary breast cancers and compare them to adjacent normal breast cells. In addition, we screened the responses of these cells with different cell surface receptors against \u003cem\u003eCrocin\u003c/em\u003e to compare and figure out the possible differences in the mechanism of action in different breast tumor types.\u003c/p\u003e"},{"header":"Materials \u0026 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCrocin\u003c/em\u003e was isolated and purified from Iranian saffron \u003cem\u003e(Crocus Sativus\u003c/em\u003e L.) by the previously registered method in our Lab. (National Patent #54958, Oct. 27, 2008). All materials were of analytical grade and prepared by Sigma-Aldrich or Merck Co.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMethods\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eBreast tissue biopsies were obtained from the tumor breast tissue after surgery of breast cancer patients using the approved protocol of the Ethics Committee of Tarbiat Modares University (36D-1392-02-17). Then, cancer and normal epithelial cells were isolated and cultured in the medium as described previously [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eViability assay and flow cytometry\u003c/h2\u003e \u003cp\u003eThe MTT assay was performed in the presence or absence of \u003cem\u003eCrocin\u003c/em\u003e, as described previously [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe isolated epithelial cancer cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e) were plated and then treated with \u003cem\u003eCrocin\u0026rsquo;s\u003c/em\u003e IC50 values at various intervals. Then, \u003cem\u003eCrocin\u003c/em\u003e\u0026rsquo;s extent of apoptosis induction on the cancer epithelial cells was evaluated by fluorescence-activated cell sorting (FACS) of cells stained with Annexin V- PI [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFlow cytometry analysis was also performed on all \u003cem\u003eCrocin\u003c/em\u003e-treated and untreated primary breast cancer cells to determine the EpCAM expression at the cell surface. The relative percentages of cells in the G1, S, or G2/M phases of cell cycle were analyzed by flow cytometry and calculated using FlowJo [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRT-PCR\u003c/h2\u003e \u003cp\u003eTotal pure RNA was isolated from the \u003cem\u003eCrocin\u003c/em\u003e-treated and untreated cancer epithelial cells using a RiboExTM kit (GeneAll\u0026reg;, Korea). The corresponding gene-specific mRNA primer pair sets are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe name, DNA sequence, length and melting temperature (Tm) of all the designed and used forward (F) and reverse (R) primers in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLength (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrimer Sequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTm (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHPRT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF 5\u0026rsquo;-CCTGGCGTCGTGATTAGTG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR 5\u0026rsquo;-TCAGTCCTGTCCATAATTAGTCC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.3\u003c/p\u003e \u003cp\u003e57.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCaspase-9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF 5\u0026rsquo;-CCACACCCAGTGACATCTTT-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR 5\u0026rsquo;-AAACAGCATTAGCGACCC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53.9\u003c/p\u003e \u003cp\u003e58.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ep27\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF 5\u0026rsquo;-CAAACGTAAACAGCTCGAAT-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR 5\u0026rsquo;-CATAATGCTACATCCAACGCT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57.4\u003c/p\u003e \u003cp\u003e54.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eXBP1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003euXBP1:283\u003c/p\u003e \u003cp\u003esXBP1:257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF 5\u0026rsquo;-TTACGAGAGAAAACTCATGGCC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eR 5\u0026rsquo;-GGGTCCAAGTTGTCCAGAATGC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.4\u003c/p\u003e \u003cp\u003e61.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eWestern blotting using the method described previously [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] investigated the expression of procaspase-9 (pCas9), cleaved caspase-9 (cCas9), p27, CXCR4, XBP1s, LC3II, Rap-1α, Lamin B, and β-Actin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe results of three independent experiments are reported as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, and were statistically evaluated using repeated measures and ANOVA followed by Tukey\u0026rsquo;s Post Hoc analysis between different groups and at different intervals, using SPSS version 16.0. Comparisons were considered statistically significant at a 95% confidence interval, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eThe effect of\u003c/b\u003e \u003cb\u003eCrocin\u003c/b\u003e \u003cb\u003eon the viability of normal and cancer primary epithelial cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe cytotoxicity and anti-proliferative effect of varying \u003cem\u003eCrocin\u003c/em\u003e concentrations in primary normal (N) and cancer (C) epithelial cells were studied using MTT assay at different intervals (12, 24, 48 and 72 h). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, C1 to C5, and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e indicate a variation in \u003cem\u003eCrocin\u0026rsquo;s\u003c/em\u003e half inhibitory concentrations (IC50) in five isolated breast cancer cells at different times. It was between 2.56 and 4.6 mM after 24 h. At the same condition, the IC50 of \u003cem\u003eCrocin\u003c/em\u003e in these normal epithelial cells was between 5.7 and 7.2 mM after 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, N1 to N5 and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e), about two times more than those observed in the related cancer cells. The IC50 value of \u003cem\u003eCrocin\u003c/em\u003e in each cancer epithelial cell was used for further experiments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe pathoclinical details of isolated breast cancer cells.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical parameter\u003c/p\u003e \u003cp\u003eIsolated Cells\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eER\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHER2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTumor Grade\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLymph node status\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIC50 values of \u003cem\u003eCrocin\u003c/em\u003e for cancer and normal epithelial cells isolated from breast tumor tissues, after 24 h treatment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIndividual\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCrocin\u003c/em\u003e IC50 (mM)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCancerous\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCrocin\u003c/b\u003e \u003cb\u003epromotes apoptosis and induces stress in primary cancer cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe primary cancer cells were treated with their IC50 value of \u003cem\u003eCrocin\u003c/em\u003e for up to 24 h and then were analyzed by flow cytometry. The results showed increased Annexin V positive cell populations (both early and late apoptotic cells) from 6 to 24 h. Because of the similarity in these data in all cancer cells, the data of only one isolated cancer epithelial cell is shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA to \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD at different time intervals. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE shows the histogram of the necrotic, early, and late apoptotic cell percentages. Although the population of both early and late apoptotic cells was significantly higher after 24 h, there were significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) higher levels of late apoptotic cells than other populations after 24 h of \u003cem\u003eCrocin\u003c/em\u003e treatment compared to control cells. A similar pattern was observed for all other primary cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen, we evaluated the caspase-9 (Cas9) level, at mRNA and protein levels, as a marker of the intrinsic/ mitochondrial pathway of apoptosis. Figures\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB show \u003cem\u003eCrocin\u003c/em\u003e increased \u003cem\u003eCAS9\u003c/em\u003e mRNA expression up to 24 h (\u003cem\u003ep\u003c/em\u003e \u0026le; 0.001) compared to the control, but most changes were observed at 6 h (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) in all five cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC indicates the \u003cem\u003eCrocin\u003c/em\u003e-induced expression of procaspase-9 (pCas9) and activation (cleaved caspase9/ cCas9) in a time-dependent manner. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD indicates the significant induction of pCas9 expression (\u003cem\u003ep\u003c/em\u003e \u0026le; 0.01) after 6 h of \u003cem\u003eCrocin\u003c/em\u003e treatment compared with the controls in all five cancer cells. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE shows a significant increase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) in the cCas9, indicating increased Cas9 activity in the \u003cem\u003eCrocin\u003c/em\u003e-treated cells compared with the control.\u003c/p\u003e \u003cp\u003eAlthough significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) activation of Cas9 was observed after 6 h of \u003cem\u003eCrocin\u003c/em\u003e treatment in C1 and C2 (Group 1 cells), it was significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) increased in C3, C4, and C5 (Group 2) after 12 h of \u003cem\u003eCrocin\u003c/em\u003e treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, cCas9/ pCas9 ratio, clearly indicates the reverse trend of changes in this parameter by time in Group 1 versus Group 2 cells. The results show more increase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) at 6 h, then a decrease in Group 1, and a time-dependent increase up to 24 h in Group 2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) compared to the control.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eXBP1\u003c/em\u003e splicing was studied at different time intervals. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA indicates the \u003cem\u003eCrocin\u003c/em\u003e-induced splicing of \u003cem\u003eXBP1\u003c/em\u003e at the mRNA level by time. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB shows the significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) changes in the ratio of spliced \u003cem\u003eXBP1\u003c/em\u003e (\u003cem\u003eXBP1s\u003c/em\u003e) to the unspliced \u003cem\u003eXBP1\u003c/em\u003e (\u003cem\u003eXBP1u\u003c/em\u003e) mRNA. Similar changes were also observed in the protein level. Figures\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC shows the Western blot data of some proteins, including the spliced form of XBP1 (XBP1s). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD indicates a significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) increase in the XBP1s by time, especially after 12 to 24 h.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC indicates the overall accumulation of LC3-II in cancer cells. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE indicates a significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) increase in the LC3-II/ LC3-I ratio after 24 h of \u003cem\u003eCrocin\u003c/em\u003e treatment compared to untreated control cells in all five isolated cancer cells. Like the results of Cas9 activation, increasing the LC3-II/ LC3-I ratio was seen in C1 and C2 (Group 1) cancer cells after 6 and 12 h of \u003cem\u003eCrocin\u003c/em\u003e treatment, respectively. In comparison, it was increased up to 24 h in three other cells (Group 2).\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC also shows the Western blot pattern of Lamin B expression and farnesylation in the mentioned cells. The bands related to unfarnesylated Lamin B were only seen in C1 cells. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF shows that Lamin B expression in C1 decreased at 6 h and then increased up to 24 h. In addition, the unfarnesylated Lamin B was increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) up to 24 h of \u003cem\u003eCrocin\u003c/em\u003e treatment. The ratios of unfarnesylated (uF)/ farnesylated (F) Lamin B are shown in the inset of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF. Except C1, a relatively similar pattern was observed in the Lamin B expression in four other breast cancer cells (C2 to C5), which their histogram is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG. Although the level of Lamin B was different before \u003cem\u003eCrocin\u003c/em\u003e treatment (time 0), it was leveled off by time (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) up to 24 h in these cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRegulation of the cell progression and metastasis prevention by\u003c/b\u003e \u003cb\u003eCrocin\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate whether cell cycle arrest is also involved in the growth inhibitory effect of \u003cem\u003eCrocin\u003c/em\u003e, we evaluated the cell cycle distribution by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB indicate that the population of cells at the S phase significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) decreased after 12 h of \u003cem\u003eCrocin\u003c/em\u003e treatment, in all cancer cells except C1. After 24 h of \u003cem\u003eCrocin\u003c/em\u003e treatment, the percentages of sub-G1 (apoptotic) cells significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) in all isolated cancer cells. So, over 50% of the C1 and C2 cell populations were in the sub-G1 phase after 24 h. These changes were accompanied by a significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005) reduction of all cell accumulation in the G0/G1 phase after 24 h. These results suggest that \u003cem\u003eCrocin\u003c/em\u003e induces G0/G1- and S-phase cell cycle arrest in the isolated breast cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsequently, we evaluated the effect of \u003cem\u003eCrocin\u003c/em\u003e on the expression of p27, a cyclin-dependent kinase inhibitor involved in the cell cycle arrest at the G1 phase (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Although the expression of p27 mRNA was increased significantly in C1 and C2 cancer cells, after 6 h of \u003cem\u003eCrocin\u003c/em\u003e treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000), there were no significant changes in the expression of \u003cem\u003ep27\u003c/em\u003e mRNA in Group 2 (C3, C4, and C5) cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of p27 protein expression are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD. In contrast to the unchanged \u003cem\u003ep27\u003c/em\u003e mRNA levels in these cells, the level of p27 protein significantly increased (\u003cem\u003ep\u003c/em\u003e \u0026le; 0.001) in Group 2 cancer cells up to 24 h of \u003cem\u003eCrocin\u003c/em\u003e treatment. On the other hand, a significant increase in the p27 mRNA expression at 6 h in C1 and C2 cancer cells did not translate to such a large protein production.\u003c/p\u003e \u003cp\u003eWe also examined the expression of the CXCR4 protein involved in cancer progression and metastasis. Figures\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE indicate significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) and time-dependent decrease, in the CXCR4 levels, especially after 24 h of \u003cem\u003eCrocin\u003c/em\u003e treatment.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC shows the effect of \u003cem\u003eCrocin\u003c/em\u003e on the accumulation of unprenylated (uP)-Rap-1α up to 24 h after treatment. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF shows the histograms of Rap-1α expression in these cells. It indicates a similar pattern in all five cells and increased accumulation of unprenylated uP-Rap-1α in the \u003cem\u003eCrocin\u003c/em\u003e-treated cells compared to the control. However, the unprenylated form of this protein was significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in Group 2 cells up to 24 h than in Group 1 cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eWe also monitored the effect of \u003cem\u003eCrocin\u003c/em\u003e on EpCAM expression, another marker for cell growth and metastasis. Figures\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB showed that \u003cem\u003eCrocin\u003c/em\u003e treatment decreased the Ep-CAM expression after 24 h in all \u003cem\u003eCrocin\u003c/em\u003e-treated breast cancer cells compared to the control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe investigated the mechanism(s) of the anticancer effect of \u003cem\u003eCrocin\u003c/em\u003e in the primary breast cancer cells isolated from Iranian women [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. According to our literature survey, it is the first report related to the anticancer activity of \u003cem\u003eCrocin\u003c/em\u003e on primary breast cancer cells. Thus, the effect of \u003cem\u003eCrocin\u003c/em\u003e on both HER2/neu negative cells, Group 1 (C1 and C2), and HER2/neu positive cells, Group 2 (C3, C4, and C5), was investigated. \u003cem\u003eCrocin\u003c/em\u003e significantly inhibited the growth of these cancer epithelial cells and induced death with different IC50, possibly based on tumor grade, surface receptors, and other intrinsic/ genetic features of each cell. The mechanism of cell death induction was apoptosis through Cas9 upregulation and activation in all five isolated breast cancer cells. \u003cem\u003eCrocin\u003c/em\u003e also induced some unfolded protein response (UPR) and autophagy markers In addition, it decreased cancer cell proliferation by retaining the primary cancer cells in the sub-G1 phase and preventing them from entering the G0/G1 phase, especially in Group 1 cells that were HER2/neu negative. An overall increase in the p27 protein level was observed up to 24 h of \u003cem\u003eCrocin\u003c/em\u003e treatment. Especially in HER2/neu positive cells. \u003cem\u003eCrocin\u003c/em\u003e also decreased the expression of CXCR4 and EpCAM in these cancer cells.\u003c/p\u003e \u003cp\u003eAs mentioned in the introduction, we previously isolated and characterized cancer and normal epithelial cells from the breast cancer tumors of five patients [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. We treated these cells with \u003cem\u003eCrocin\u003c/em\u003e in the present study and determined their IC50 using an MTT assay. The tumors used in this study were categorized according to breast cancer grade and the expression of surface hormone receptors on the cells (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Group 1 (C1 and C2), were grade II breast cancer cases and HER2/neu negative. Group 2 (C3, C4, and C5), were grade III cases and HER2/neu positive. The lymph nodes were involved in all cases except C1. The grade II breast cancer cells were more sensitive to \u003cem\u003eCrocin\u003c/em\u003e than grade III cases. Cell death was induced with lower doses of this natural carotenoid in these cells than in grade II cells. We previously determined the IC50 of \u003cem\u003eCrocin\u003c/em\u003e in various breast cancer cell lines. It has obtained 2.7 mM in MDA-MB-231 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], 3.0 mM in MCF-7 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], 3.1 in MDA-MB-468 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and 3.6 mM in BT-474 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. As mentioned in our previous review paper, the IC50 of \u003cem\u003eCrocin\u003c/em\u003e has been reported up to 5.5 mM in various cancer cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the other hand, our previous \u003cem\u003ein vivo\u003c/em\u003e studies indicated that four-weeklies i.p. injection of 150 mg/Kg body weight \u003cem\u003eCrocin\u003c/em\u003e efficiently removed tumors or reduced tumor sizes in the NMU-induced breast cancer in rats [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and in the 4T1-induced breast cancer in BALB/c mice [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The \u003cem\u003ein vitro\u003c/em\u003e colony-formation assay also indicated the effectiveness of \u003cem\u003eCrocin\u003c/em\u003e at \u0026micro;M concentration in breast cancer cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Thus, in contrast to the high \u003cem\u003eCrocin\u003c/em\u003e concentrations needed to induce apoptosis in various cancer cells, the \u003cem\u003ein vivo\u003c/em\u003e dose needed to suppress the tumor growth is significantly low.\u003c/p\u003e \u003cp\u003eAlthough the safety of \u003cem\u003eCrocin\u003c/em\u003e has been reported for human [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and in normal animals [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], its effect on normal epithelial breast cell has not been reported. In some studies, MCF-10 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], which is not a normal breast cell, and in some others, fibroblast cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] with entirely different genetic patterns from the breast epithelial cells have been used. Thus, in the present study, we applied the primary breast normal cells isolated from the adjacent tissues of tumors to compare the results. The data indicated no toxicity of \u003cem\u003eCrocin\u003c/em\u003e for normal breast cells of the same tissue at the toxic dosages obtained for those cancer cells. Cancer cells were significantly more susceptible to \u003cem\u003eCrocin\u003c/em\u003e treatment than normal cells.\u003c/p\u003e \u003cp\u003eContinuing the mechanistic study, we reported a \u003cem\u003eCrocin\u003c/em\u003e-induced Cas9 expression in five isolated breast cancer cells. Additionally, \u003cem\u003eCrocin\u003c/em\u003e induced the cleavage of pCas9 and the production of cCas9 in these cancer cells. These results agree with previous studies showing the apoptotic effect of \u003cem\u003eCrocin\u003c/em\u003e in various breast cancer cell lines [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and the role of mitochondria-mediated apoptosis, which is precisely regulated by the Bcl-2 family proteins [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Furthermore, the inhibitory role of \u003cem\u003eCrocin\u003c/em\u003e on the growth of other human cancer cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and in animal models of breast and gastric cancers [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] could be attributed to a similar mechanism.\u003c/p\u003e \u003cp\u003eAnother method to approve apoptotic cell death is to detect and quantify the percentages of apoptotic or necrotic cells by annexin V/ PI staining using flow cytometry [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Our results showed a similar apoptotic pattern in all \u003cem\u003eCrocin\u003c/em\u003e-treated isolated breast cancer cells, with significantly higher populations of apoptotic cells than necrotic cells.\u003c/p\u003e \u003cp\u003eActivation of the UPR as a result of the endoplasmic reticulum (ER) stress has a crucial role in protein homeostasis and other diverse functions involved in the process of breast cancer progression. In this regard, we studied the impact of \u003cem\u003eCrocin\u003c/em\u003e on \u003cem\u003eXBP1 slicing\u003c/em\u003e, one of the ER stress indicators [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. After \u003cem\u003eCrocin\u003c/em\u003e treatment, the \u003cem\u003eXBP1\u003c/em\u003e mRNA splicing increased by time, which ultimately resulted in the expression of the spliced protein (XBP1s) in all five breast cancer cells. Similar changes has been reported in MDA-MB-468 and BT-474 breast cancer cell lines after \u003cem\u003eCrocin\u003c/em\u003e treatment [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, we observed the inductive effect of \u003cem\u003eCrocin\u003c/em\u003e on LC3II production and accumulation, which was accompanied by an increase in the LC3II/LC3I ratio in all five cancer cells. We have previously shown that \u003cem\u003eCrocin\u003c/em\u003e changed the LC3II/LC3I ratio in MDA-MB-468 and MCF-7 breast cancer cell lines [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The conversion of LC3I to LC3II has been introduced as an autophagy marker [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Our data also showed a time-dependent decrease of Lamin B protein in these primary breast cancer cells after \u003cem\u003eCrocin\u003c/em\u003e treatment. It has been shown that LC3 directly interacts with Lamin B1, and then this complex bind to Lamin-associated chromatin domains. These interactions cause the autophagy-mediated destruction of the nuclear lamina. The nuclear lamina degradation impairs cell proliferation by inducing cell-cycle arrest as a tumor-suppressive mechanism [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUPR and autophagy have been known as adaptive mechanisms to regulate cellular function during stress. If the stress is prolonged, apoptotic cell death ensues [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Recently, we showed a \u003cem\u003eCrocin\u003c/em\u003e-induced ROS production in cancer cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, the process can induce UPR-regulated autophagy and apoptosis in tumor cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. A similar phenomenon, autophagic-induced apoptosis, has been reported as the mechanism of action of other anticancer compounds in other types of cancer [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs an antitumor agent, \u003cem\u003eCrocin\u003c/em\u003e induced cell cycle arrest via changing p53, p21, and cyclin D1 in the NMU-induced breast cancer in rats [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The data in the present study also indicates the cell-cycle arrest induction at the G0/G1 phase after \u003cem\u003eCrocin\u003c/em\u003e treatment in primary breast cancer cells. A similar role of \u003cem\u003eCrocin\u003c/em\u003e has also been reported in the human gastric cancer cell line [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. \u003cem\u003eCrocin\u003c/em\u003e retains cells in the sub-G1 phase and decreased their entry into S phases, especially in HER2/neu positive cells. To investigate the effect of \u003cem\u003eCrocin\u003c/em\u003e on cell cycle regulators, we examined the expression of p27. Similar to the present study, a reverse relation between p27 and HER2/neu expression has been shown in breast cancer [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. HER2/neu signals caused a decrease in p27 stability and enhancement of its degradation. By blocking HER2/neu, p27 has been upregulated [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. So, in C1 and C2 cancer cells (grade II and HER2/neu negative), the p27 mRNA expression was higher than in Group 2 cancer cells (grade III and HER2/neu positive). Although \u003cem\u003eCrocin\u003c/em\u003e treatment in HER2/neu positive cells did not significantly alter p27 mRNA levels, the protein levels of p27 increased in these cancer cells. This effect may be due to \u003cem\u003eCrocin\u003c/em\u003e\u0026rsquo;s ability to overcome the HER2/neu or its downstream signaling pathway and inhibition of p27 degradation.\u003c/p\u003e \u003cp\u003eAll primary breast cancer cells in this study were isolated from patients with ductal carcinoma in situ. As the data show, EpCAM was expressed in all of them. EpCAM has been known to be overexpressed in epithelial cancer cells, and its overexpression appears to be associated with enhanced proliferation and malignant potential [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. \u003cem\u003eCrocin\u003c/em\u003e inhibited the EpCAM expression and was more effective in grade II tumor cells than in grade III. It is conceivable that HER2/neu-independent mechanisms may be responsible for the downregulation of EpCAM in \u003cem\u003eCrocin\u003c/em\u003e-treated breast cancer cells.\u003c/p\u003e \u003cp\u003eA link between uP-Rap-1α and cell proliferation and tumor cell migration and invasion has been extensively studied and reviewed [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Furthermore, the diminished Rap-1α expression decreased cell migration ability [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Our data also indicated the increased uP-Rap-1α due to the \u003cem\u003eCrocin\u003c/em\u003e treatment of all breast cancer cells. However, the changes were different in Group 1 and Group 2 cancer cells. Before \u003cem\u003eCrocin\u003c/em\u003e treatment, the accumulation of uP-Rap-1α in Group 2 cancer cells was significantly lower than in Group 1. \u003cem\u003eCrocin\u003c/em\u003e induced the accumulation of uP-Rap-1α more than 4-fold in Group 2 cancer cells compared with Group 1, which was increased less than 1-fold. So, after 12 h of \u003cem\u003eCrocin\u003c/em\u003e treatment, all five cancer cells reached the same level of uP-Rap-1 accumulation.\u003c/p\u003e \u003cp\u003eA significant correlation between HER2 and CXCR4 expression has been observed in human breast tumor tissues, related to cancer recurrence, metastasis, and poor survival rates [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Furthermore, the degree of CXCR4 expression, a chemokine signaling system important in breast cancer progression and metastasis [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], was also higher in HER2/neu positive (Group 2) than HER2/neu negative (Group 1) cancer cells. Here, we observed that \u003cem\u003eCrocin\u003c/em\u003e significantly decreased the expression of CXCR4 in all five breast cancer cells. However, it was more effective in the HER2/neu positive group than the HER2/neu negative cells. It indicates that \u003cem\u003eCrocin\u003c/em\u003e might inhibit the HER2/neu signaling and its association with metastasis in breast cancer cells.\u003c/p\u003e \u003cp\u003eThe limitations of this study include the limited number of primary breast cells. Thus, it should continue to use a larger population of primary breast cancer cells with different genetic characteristics. Furthermore, a clinical trial should be designed for \u003cem\u003eCrocin\u003c/em\u003e application as a supplement in breast cancer patients. However, it shows that tumor genetics is essential in the \u003cem\u003eCrocin\u003c/em\u003e\u0026rsquo;s anticancer mechanism(s). Therefore, this subject is crucial in precision medicine and should be considered in future studies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the present study, XBP1 splicing and LC3-II accumulation, MTT assay, flow cytometry data, and the pCas9 overexpression and activation confirmed that \u003cem\u003eCrocin\u003c/em\u003e could induce stress in cancer cells. When the stress was prolonged, apoptotic cell death occurred in a time-dependent manner in all five breast cancer cells. LC3-II accumulation, Lamin B expression, and prenylation were also investigated as the factors involved in the autophagy-mediated destruction of the nuclear lamina.\u003c/p\u003e \u003cp\u003eThis study highlighted the importance of breast cancer cell characteristics and response to treatment. Since the cells were genetically different, different doses of \u003cem\u003eCrocin\u003c/em\u003e induced death in the isolated cancer cells through two different mechanisms of apoptosis and cell cycle arrest. It also decreased the expression of proteins involved in proliferation and metastasis in breast cancer cells isolated from biopsies with HER2/neu positive rather than HER2/neu negative. The effect of \u003cem\u003eCrocin\u003c/em\u003e depends explicitly on the proteins of HER2/neu and its downstream signaling involved in proliferation and metastasis. Thus, by studying each patient\u0026rsquo;s biopsy and investigating the genetic/ protein patterns, we can determine the amount of \u003cem\u003eCrocin\u003c/em\u003e needed to induce stress and, finally, apoptosis and activate different mechanisms for cell death induction in tumor cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eElite Researcher Grant Committee of the National Institute for Medical Research Development (NIMAD) supported the research reported in this publication under award numbers [971400], Tehran, Iran.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.Z.B.: Supervised the study; Conception and design of the work; Analysis; Interpretation of data, Editing and Revising the manuscript.N.F.: Conception and Design; Data acquisition, Analysis; Interpretation of data; Drafting the manuscript.H.H.: Study Design, Western blotting, and Data Analysis.S.A.H.: Study Design, Data acquisition and Analysis.S.A.: Conception and Design, Data interpretation, Editing the manuscript.F.T.: Conception and Design, Data interpretation, Editing the manuscript.M.-A.M.: Conception and Design, Clinical Control; manuscript evaluation.All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank the \u0026ldquo;Iran Science Elites Federation\u0026rdquo; for supporting Professor Bathaie and granting Dr. Faridi a Postdoc.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw data for Western blot analysis will be sent for review if requested.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGlobal Breast Cancer Initiative Implementation Framework (2023) assessing, strengthening and scaling-up of services for the early detection and management of breast cancer. 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Cancer Lett 238(1):30\u0026ndash;41\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Breast Cancer, Apoptosis, Stress Response, LC3-II Accumulation, Metastatic Markers, EpCAM","lastPublishedDoi":"10.21203/rs.3.rs-4711052/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4711052/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThe anticancer effect of \u003cem\u003eCrocin\u003c/em\u003e, a natural C20 carotenoid, has been previously demonstrated in different cancer cell lines and animal cancer models. Herein, we investigated its effect on primary breast cancer cells isolated from women\u0026rsquo;s breast tumor samples.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe previously isolated and characterized epithelial breast cancer and normal cells from female patients. In this study, we treated five cancer cells and five normal cells from the same sample with \u003cem\u003eCrocin.\u003c/em\u003e Then, the type and mechanisms of \u003cem\u003eCrocin\u003c/em\u003e-induced cell death were studied using different techniques.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAll of these tumors were estrogen and progesterone receptor-positive. Two samples were in grade II and HER2-negative, while three others were grade III and HER2-positive. The IC50 of \u003cem\u003eCrocin\u003c/em\u003e were obtained using MTT assay for all cells. It induced procaspase-9 expression and cleavage, sub-G1 accumulation, XBP1 mRNA splicing and expression of the spliced XBP1, LC3-II accumulation, and accumulation of unprenylated Rap1α in all cancer cells. The p27 mRNA expression was only induced in cells isolated from HER2-negative samples. However, an increase in the p27 protein level was observed in all cells. \u003cem\u003eCrocin\u003c/em\u003e also down-regulated the CXCR-4 and suppressed EpCAM in these cancer cells. The unfarnesylated Lamin B was observed only in one sample.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCrocin\u003c/em\u003e suppressed the proliferation of human primary epithelial breast cancer cells, enhanced stress responses, and decreased metastatic markers. There was a difference between p27 expression in HER2-negative and positive tumors.\u003c/p\u003e","manuscriptTitle":"Crocin Induces Apoptosis in Primary Cancer Epithelial Cells Isolated from Human Breast Tumors via Different Mechanisms in HER2- Negative or Positive Cells: A Preliminary Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-10 12:19:07","doi":"10.21203/rs.3.rs-4711052/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2f99f387-08bc-4276-a186-22017713da31","owner":[],"postedDate":"August 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-13T14:22:50+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-10 12:19:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4711052","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4711052","identity":"rs-4711052","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}