Silencing of the CrkL gene reverses the doxorubicin resistance of K562/ADR cells through regulating PI3K/Akt/MRP1 signaling

Abstract Background Doxorubicin is a first‐line chemotherapy agent on human myelogenous leukemia clinical treatment, but the development of chemoresistance has largely limited curative effect. In this study, we aimed to evaluate the biological function and molecular mechanisms of CrkL to Doxorubicin resistance. Methods Quantitative reverse transcription‐PCR (qRT‐PCR) assay was performed to examine the expression of CrkL in K562 and K562/ADR cells. The expression of CrkL was silenced through RNA interference technology. MTT assay and flow cytometry were performed to detect the proliferation inhibition and apoptosis rate after CrkL siRNA transfection. The protein expression changes of PI3K/AKT/MRP1 pathway induced by CrkL siRNA were observed by Western Blot assay. Xenograft tumor model was carried out to observe tumor growth in vivo. Results We observed that silencing of CrkL could effectively increase apoptosis rate induced by doxorubicin and dramatically reversed doxorubicin resistance in K562/ADR cells. Further studies revealed knockdown CrkL expression suppressed PI3K/Akt/MRP1 signaling, which indicated CrkL siRNA reversed doxorubicin effect through regulating PI3K/Akt/MRP1 pathway. In addition, overexpression of MRP1 could evidently reduce apoptosis rate and reversed the inhibitory effects of doxorubicin resistance caused by CrkL siRNA on K562/ADR cells. Finally, in vivo experiments revealed that CrkL silencing acted a tumor‐suppressing role in myelogenous leukemia via regulating PI3K/Akt/MRP1 signaling. Conclusion Together, we indicated that CrkL is up‐regulated in myelogenous leukemia cells and silencing of CrkL could reverse Doxorubicin resistance effectively. These results show a potential novel strategy for intervention chemoresistance in myelogenous leukemia during chemotherapy.


| INTRODUC TI ON
Leukemia is one of most frequent types of human cancers worldwide. It can be divided into acute and chronic leukemia according to the degree of differentiation and the length of the natural course.
On the basis of the types of blood cells affected, it can be divided into myeloid and lymphoblastic leukemia. 1 Chemotherapy is the crucial approach to treat leukemia clinically and Doxorubicin (Dox) is the most commonly used chemo-therapeutic agent. 2 Dox is an anthracycline-based anti-tumor drug and kills leukemia cells by inhibiting DNA transcription and RNA synthesis, and also destroys the DNA and protein structures of leukemia cells. 3 But the occurrence of chemoresistance and side effects seriously impacts the treatment efficacy and shortens patients' survival time. Therefore, a great deal of attention has been focused on identifying the genetic and molecular mechanisms of Dox resistance. Until now, series of chemoresistance research revealed the ATP-binding cassette (ABC) transporter superfamily contributed to multi-drug resistance(MDR), 4 which contained the P-glycoprotein (P-gp),MRP1(multi-drug-associated resistance protein 1) and ABCC1 (ATP-Binding Cassette Subfamily C Member 1) transporter. MRP1 utilizes the energy from ATP-binding or hydrolysis and actively effluxes intracellular drug out of cells leading to MDR. 5,6 CrkL is a member of the Crk adapter protein family, which is involved in two alternatively spliced isoforms of CRK(Crk I and Crk II), human homolog of avian sarcoma retroviral v-crk oncogene and Crk-like protein (CrkL). 7 Recent studies have demonstrated that CrkL acts a pivotal part in cell proliferation and associated with the migration and invasion in range of cancers such as bladder, 8 breast cancers, 9 lung, 10 stomach, 11 and liver. 12 The high expression level of CrkL has been related to advanced-stage cancers with highgrade aggressiveness and poor prognosis. Cheung et al. 10 found that overexpression of CRKL triggered gefitinib resistance in epidermal growth factor receptor (EGFR)-mutant cells via actuating AKT signaling and extracellular signal-regulated kinase in non-small cell lung cancer. The high expression of CrkL intervened CCL20/ CCR6-induced EMT through Akt signaling to the occurrence and development of gastric cancer. 13 The studies elucidated that CrkL contributed to the amplification and initiation of oncogenic signals to promote tumorigenesis. However, the function and molecular mechanism underlying CrkL expression in non-solid malignancies are largely undiscovered.
In the present study, the main objective was to determine the biological function of CrkL on Dox resistance of myelogenous leukemia and to find the molecular mechanism by which it occurs. We found that silencing CrkL expression reversed the

| Cell transfection
The siRNA oligonucleotide targeting the human CrkL gene (sense:

| Apoptosis assay
Cell apoptosis assay was carried out using AnnexinⅤ-FITC/PI Kit according to the manufacturer's instructions. Briefly, cells were treated for 48 h and harvested. After washing three time with precooled PBS, the cells were resuspended in 0.5 mL binding buffer.
And then 10 µL propidium iodide and 10 µL Annexin-V-FITC were further added into the system for 15 min incubation. Finally, the cells were washed to remove excess stains and detected via the flow cytometer (BD Biosciences).

| Quantitative real-time PCR assay
Total RNA was extracted using Trizol reagent (Invitrogen) and then reversely transcribed to cDNA using a PrimeScript RT Reagent kit (Takara). 25 μL reaction mixture was consisted with 12.5 μL of SYBR FAST master mix, 1 μL aliquot of aptamer-containing solution, 0.5 μL of reverse primer and nuclease-free water. qRT-PCR was carried out to initial denaturation for 2 min at 95°C followed by cycling at 95°C for 30 s, and then annealing/extension for 20 s at 72°C. This process was repeated on PCR machine for 40 times. Data were analyzed using the comparative Ct method (2−ΔΔCt). The primers used for real-time PCR were as listed in Table 1.

| Western blot analysis
Cells were washed in cold PBS buffer for three times and lysed in ice-cold RIPA buffer for 30 min. Cell debris was removed through centrifugation at 12,000 rpm for 10 min at 4°C. The protein concentration was verified via the BCA protein quantitation kit. Protein samples were electrophoresed on SDS-PAGE and then transferred to PVDF membranes. 5% skim milk was used to block the nonspecific binding at room temperature for 2 h. The membranes were immunoblotted with primary monoclonal antibodies against CrkL

| Immunohistochemistry
The tumor tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated in a graded alcohol series and embedded in paraffin followed by cutting into 5 μm sections. The sections were deparaffinized, rehydrated with a graded alcohol series, and then incubated in 96℃ with 0.01 mol/L sodium citrate buffer for the antigen retrieval. After incubation in 5% H 2 O 2 for 2 h, the sections were incubated with Ki67 primary antibodies overnight at 4℃. Immunostaining was performed using streptavidin-peroxidase and diaminobenzidine (DAB) according to the manufacturer's instructions (Beyotime). Finally, the sections were observed under a fluorescence microscope (Leica) and pictured.

| Statistical analysis
Statistical analyses were carried out using the SPSS 19.0. Continuous variables were expressed as the mean ± standard deviation from at least three separate experiments. Differences were analyzed by Student's t test between two groups or one-way analysis of variance (ANOVA) among multiple groups. p values <0.05 were considered statistically significant.
The CrkL siRNA2, which showed the greatest interference effect, was selected for follow-up experiments.

| CrkL silencing reduced the doxorubicin resistance and increased apoptosis in K562/ADR cells
We examined whether CrkL silencing sensitizes K562/ADR cells to  (Table 2).
Subsequently, flow cytometry analysis was performed to further determine the effect of CrkL on Dox-induced apoptosis. The results revealed that CrkL silencing dramatically enhanced Dox-induced apoptosis in K562/ADR cells (Figure 2A-B). Collectively, silencing of CrkL improved Dox sensitivity in K562/ADR cell.

MRP1 has been reported to play an essential role in chemoresist-
ance. In this study, to further determine that CrkL affects Dox

| CrkL silencing suppressed the tumor growth of K562/ADR cells in vivo
Next, we carried out in vivo study to further investigate the biological effects of CrkL silencing in the progression of myelogenous leukemia. Stable K562/ADR cell line was established with transfection of CrkL knock down vectors and its control vector respectively. As the results showed, the tumor growth was inhibited by CrkL knockdown ( Figure 5A-B). And the weight of the tumors was reduced by CrkL siRNA treatment ( Figure 5C). Ki67 is a nuclear protein associated with tumor cell proliferation and used widely as a marker to determine the tumor growth in routine pathological investigation. 15 The IHC staining was applied to determine the Ki67 expression in tumor tissues and the results showed CrkL silencing could notably inhibit Ki67 expression compared with the vector group ( Figure 5D-E). Furthermore, Western Blot assay revealed that the expression level of p-AKT, CrkL and MRP1 protein levels inhibited while AKT expression remained unchanged compared with the NC groups after CrkL siRNA treatment ( Figure 5F-G). Thus, we concluded that CrkL silencing inhibited the promotion of myelogenous leukemia in vitro and in vivo.

| DISCUSS ION
Human myelogenous leukemia is one of most common malignancies worldwide with characteristics of rapid progression, poor prognosis and high mortality. Chemotherapy is the primary clinical treatment.
But the occurrence of drug resistance is the main obstacle during the long-time treatment, which leads to less than expected curative effect. 16 Therefore, it has grown attention on exploring the molecular and reverse the chemoresistance in colon carcinoma cells. 21 CrkL is an adaptor protein involved of a single N-terminal Src homology 2 (SH2) domain followed by two SH3 domains. 22 The SH2 domain binds to a polyproline (PPII) peptides of pTyr-x-x-Pro motif, while the SH3N domain intervenes the targets through a prolinerich Pro-x-x-Pro-x-(Lys, Arg) motif. 23,24 The high expression of CrkL improves adhesion-independent proliferation in fibroblasts 25   CrkL silencing in vivo significantly suppressed p-AKT, CrkL, MRP1 protein levels while AKT expression remained unchanged compared with the control and NC groups. The data were represented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ns means not significant, p > 0.05 In conclusion, the results of this study supply evidence in support of utilizing siRNAs as a molecularly therapeutic method for Dox resistance to the treatment of human myelogenous leukemia, and further studies are warranted to deep understand the mechanisms of drug resistance in human myelogenous leukemia.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest to declare in this research.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data, models and code generated or used during the study appear in the submitted article.