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

doi:10.21203/rs.3.rs-4711052/v1

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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

https://doi.org/10.21203/rs.3.rs-4711052/v1

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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

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

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 (1×10⁵) 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.

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)
Individual	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. 2A to 2D at different time intervals. Figure 2E 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 3A and 3B 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 3C indicates the Crocin-induced expression of procaspase-9 (pCas9) and activation (cleaved caspase9/ cCas9) in a time-dependent manner. Figure 3D 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 3E 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. 3E).

Figure 3F, 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 4A indicates the Crocin-induced splicing of XBP1 at the mRNA level by time. Figure 4B 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 4C shows the Western blot data of some proteins, including the spliced form of XBP1 (XBP1s). Figure 4D indicates a significant (p = 0.000) increase in the XBP1s by time, especially after 12 to 24 h.

Figure 4C indicates the overall accumulation of LC3-II in cancer cells. Figure 4E 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 4C 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 4F 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. 4F. 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. 4G. 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. 5A and 5B 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. 6A and 6D). 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. 6C and 6D. 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 6C and 6E indicate significant (p = 0.000) and time-dependent decrease, in the CXCR4 levels, especially after 24 h of Crocin treatment.

Figure 6C shows the effect of Crocin on the accumulation of unprenylated (uP)-Rap-1α up to 24 h after treatment. Figure 6F 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 7A and 7B showed that Crocin treatment decreased the Ep-CAM expression after 24 h in all Crocin-treated breast cancer cells compared to the control.

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.

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.

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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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

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