Heme oxygenase‐1 induction mediates chemoresistance of breast cancer cells to pharmorubicin by promoting autophagy via PI3K/Akt pathway

Abstract Background Concerns about breast cancer had become the most dangerous cancer to women over the world, more and more anti‐cancer agents are developed to treat this malignancy. Pharmorubicin is a cytotoxic drug, widely used in the treatment of breast cancer, but its role is limited because of chemoresistance produced by cells. This study focused on exploring the influence of autophagy on the resistance of pharmorubicin in breast cancer cells. Methods The cell survival of breast cancer cells was detected by MTT. The mRNA expression of heme oxygenase‐1 (HO‐1) was tested by qRT‐PCR. The protein expression of HO‐1, autophagic proteins (LC3‐I,LC3‐II and Beclin‐1), PI3K and Akt was detected by Western blot. Cell autophagy was examined by Cyto‐ID Autophagy Detection Kit. Results After being treated with pharmorubicin, the expression of HO‐1 and autophagy related proteins was significantly enhanced, but the cell survival ratio in the two cell lines decreased. After autophagy was inhibited, HO‐1 expression in two cells was down‐regulated. When pharmorubicin‐resistant cells were transfected with si‐HO‐1, the cell survival decreased and the protein expression of HO‐1, autophagic proteins (LC3‐II/LC3‐I and Beclin‐1) as well as autophagy were all down‐regulated, while in pharmorubicin‐resistant cells transfected with pcDNA3.1‐HO‐1, the results were reverse. When the PI3K or Akt was inhibited, PI3K, p‐Akt, HO‐1, autophagic proteins and autophagy were decreased remarkably. Conclusion It was proved that HO‐1 induction mediated chemoresistance of pharmorubicin in breast cancer cells by promoting autophagy via PI3K/Akt pathway.

therapy for breast cancer, and it can be used alone or combined with chemotherapy to reduce disease progression and has more favourable impact on survival of patients. Chemoresistance is considered a major factor influencing breast cancer clinical outcomes. However, current progress in finding a potent, selective method to overcome cancer drug resistance has become an urgent issue around the world. 1 Heme oxygenase (HO) which is the limited enzyme in heme degradation catalyses the oxidation of heme to produce some biological active molecules: carbon monoxide, ferrous ion and biliverdin.
HO has 2 isozymes, heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2), and HO-1 is high inductivity under various chemical and physical cellular stresses. Given that HO-1 has cytoprotective properties, it has attracted great attention on promoting tumour cell survival in many cancers as followings: increased expression of HO-1 promoted primary and metastatic prostate tumour growth 4 ; Up-regulating of HO-1 expression might be effective in controlling cells migration in lung cancer 5 ; HO-1 overexpression could mediate EGFinduced colon cancer cell proliferation via PI3K/Akt signalling pathway. 6 Furthermore, it was reported that the activation of Src/STAT3/ HO-1/autophagy signalling was supposed to play a crucial role in protecting breast cancer cells from doxorubicin-induced cytotoxicity. 7 These contrasting observations have undoubtedly increased the significance of HO-1 in the field of cancer biology. But there is rare research of the relationship between pharmorubicin resistance and autophagy in breast cancer.
Autophagy is regarded as the cell degradation of unnecessary or dysfunctional cell components, containing macromolecular compounds or cellular organelles, through the action of autophagosome. In normal cells, low level of autophagy could enhance recycling of cellular survival by depredating senescent organelles 2 whereas in tumour cells, the role of autophagy is more complex. 8 Ning et al found out that autophagy played a role in breast cancer resistance to chemotherapy, endocrine therapy and trastuzumab treatment. 2 In breast cancers cells, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a central role in cell growth, survival, proliferation and autophagy. 9 Some researchers showed that deregulated activity of PI3K/Akt pathway might lead to uncontrolled cell growth, survival, migration and invasion for breast cancer cells contributing to tumour formation. 10,11 But not only that, fascine was proved to play a role in the chemotherapeutic resistance of breast cancer cells predominantly by the PI3K/Akt pathway. 12 These researches showed that the PI3K/Akt pathway is closely related to the growth, chemotherapeutic resistance and autophagy of breast cancer cells.
This study would like to investigate the influence of HO-1 on the resistance of pharmorubicin in breast cancer cells, and the effect of HO-1 on cell survival and autophagy in pharmorubicin-resistant cells. Furthermore, we researched the connection between PI3K/Akt signalling pathway and breast cancer cell autophagy.

| Cell survival (MTT) assay
Cells were seeded into 96-well plates at 5000 cells per well and were treated as indicated for 12-48 hours depending on the experimental conditions. MTT (10 μL, 5 mg/mL) was added to per well and incubated for 4 hours. Finally, the medium from each well was replaced by 100 μL DMSO to dissolve the formazan before measure-

| RT-qPCR
To determine the mRNA expression of HO-1, qRT-PCR analysis was performed. Total RNA was extracted from cells by TRIzol ® (Invitrogen) and treated with DNase I (Invitrogen). cDNA was synthesized from 1 μg total RNA, using random primers with a ReverTra Ace qPCR RT Kit (Hitachi, Toyobo, Japan

| Cell autophagy analysis
The autophagy of cells was detected by Cyto-ID Autophagy Detec-

| Statistical analyses
Statistical analysis was carried out by the two-tailed Student's t test or one-way ANOVA. All analyses were performed using GraphPad

| Pharmorubicin increased HO-1 expression and autophagy in breast carcinoma cells
To

| Inhibition of pharmorubicin-induced autophagy decreased cell viability
Chloroquine is an antimalarial drug that currently approved by Food and Drug Administration to treat rheumatoid arthritis and other autoimmune diseases as an autophagy inhibitor. 17 To study the rela-

| Pharmorubicin-induced autophagy was regulated by HO-1 in breast carcinoma cells
In the further analysis, we tried to explore the downstream signalling  Autophagy is known to be connected with cancer in two contrasting mechanisms in that it is either a tumour inhibitor or a tumour protector. 2,20 The dual role of autophagy in cancer is augmented by conflicting reports about the effect of autophagy on chemotherapeutic treatment. 7 Ning et al and Yu et al found out that the suppression of autophagy might promote the drug resistance of breast cancer cell. 2,21 However, some reports showed that autophagy is induced in cancer cells as a survival strategy against these drugs. 22 For example, Zhang et al proposed that TRPC5-induced autophagy promoted drug resistance in breast carcinoma via CaMKKβ/AMPKα/mTOR pathway. 23 Similarly, the results in our study showed that the pharmorubicin resistance was up-regulated after promoting autophagy in breast cancer cells.

MDA-MB-231/EP1 and MCF-7/EPI cells
Because of these facts, it was confirmed that regulation of autophagy by HO-1 appears to be one of the most important functions to regulate the drug resistance of breast cancer cells. PI3K/Akt signalling pathway is one of the major upstream cellular signals in breast cancer cells. 2 Zhong et al found that endoplasmic reticulum stress activation could promote breast cancer cell autophagy and apoptosis and enhance chemosensitivity of MCF-7 cells by inhibiting the PI3K/AKT/mTOR signalling pathway. 19 In current study, we

DISCLOSURE
Ethics approval and consent to participate: N/A; Consent for publication: This manuscript has been approved by all authors for publication; Availability of data and material: N/A.

CONFLI CT OF INTERESTS
The authors confirm that there are no conflicts of interest.