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. 2016 Aug 30;7(35):56371-56382.
doi: 10.18632/oncotarget.10890.

Combining GRP78 suppression and MK2206-induced Akt inhibition decreases doxorubicin-induced P-glycoprotein expression and mitigates chemoresistance in human osteosarcoma

Yuan-Zheng Xia  1 , Lei Yang  1 , Gui-Min Xue  1 , Chao Zhang  1 , Chao Guo  1 , Yan-Wei Yang  1 , Shan-Shan Li  1 , Lu-Yong Zhang  1 , Qing-Long Guo  1 , Ling-Yi Kong  1
Affiliations

Combining GRP78 suppression and MK2206-induced Akt inhibition decreases doxorubicin-induced P-glycoprotein expression and mitigates chemoresistance in human osteosarcoma

Yuan-Zheng Xia et al. Oncotarget. .

Abstract

P-glycoprotein (P-gp) overexpression is associated with poor prognosis and drug-resistance in osteosarcoma (OS), but the underlying mechanisms remain incompletely understood. Here, we examined the regulation of P-gp, GRP78, and phospho-Akt in doxorubicin (DOX)-treated OS cells. DOX induced P-gp expression, which was associated with increased GRP78 levels and Akt activation in vitro and in vivo. Functional analysis showed that Akt induces P-gp and GRP78 expression, which contributes to the DOX-induced Akt activation. Examination of the relationship between Akt and GRP78 demonstrated that GRP78 suppression attenuates the Akt activity in OS parental sensitive and resistant cells, indicating that GRP78 is required for full Akt activity. Inhibition of Akt activity using MK2206 decreased GRP78 expression in OS cells, which enhanced the inhibitory effect of MK2206 on P-gp expression. GRP78 knockdown combined with MK2206 suppressed the development of DOX resistance in OS cells and inhibited the in vivo tumor growth in the presence of DOX. These results support the development of novel therapeutic strategies that target GRP78 and Akt to sensitize OS cells for chemotherapy.

Keywords: Akt; GRP78; P-gp; multidrug resistance; osteosarcoma.

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Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Figure 1
Figure 1. DOX induces P-gp, GRP78, and p-Akt expression in vitro and in vivo
(A) U-2 OS and MG-63 cells were treated with conventional chemotherapeutic drugs in the concentration of their IC50 values for OS cells for 24 hr and detected by qRT-PCR. The Δ cycle threshold method was used for the calculation of relative differences in mRNA abundance. Data were normalized to the expression of GAPDH. The results of real-time RT-PCR were expressed as fold-changes. The normalized value of the target mRNA of the control group is arbitrarily presented as 1 (n = 6). (B) U-2 OS and MG-63 cells were treated with 1 μM DOX for 0 to 72 hr. The expression of P-gp at the membrane surface and the levels of GRP78, p-Akt and Akt in the whole-cell lysates were detected by Western blot and normalized by GAPDH (n = 3). (C) Nude mice bearing xenograft tumors were treated with DOX by intraperitoneal injection once every four days. Three mice were selected randomly and sacrificed every day. Xenografts were removed, fixed and paraffin embedded. IHC staining was performed by using P-gp, GRP78 and p-Akt antibodies (×400) (n = 6). (D) Western blot results of the membrane lysates or total lysates from OS cell lines and their resistant sublines (n = 3). Data are represented as mean ± SD.
Figure 2
Figure 2. Knockdown of GRP78 slightly prevents DOX-induced P-gp expression in OS parental cell lines and resistant sublines
(A–F) OS cells and their resistant sublines were transfected transiently with siGRP78 or control siRNA for 24 hr and then treated with 1 μM DOX for 0 to 72 hr. Western blot analysis was performed with GAPDH as loading control. Data are represented as mean ± SD (n = 3).
Figure 3
Figure 3. MK2206 inhibits DOX-induced P-gp expression in OS parental cell lines and resistant sublines
(A and B) OS cells and their resistant sublines were treated with 30 nM MK2206 for 4 hr followed by treating with 1 μM DOX for 0 to 72 hr. Western blot analysis was performed with GAPDH as loading control. Data are represented as mean ± SD (n = 3).
Figure 4
Figure 4. GRP78 inhibition enhances the inhibitory effect of MK2206 on P-gp expression
(A–F) Cells were transfected transiently with siGRP78 or control siRNA for 24 hr. Subsequently, cells were incubated with 30 nM MK2206 for 4 hr followed by treating with 1 μM DOX for 0 to 72 hr. (G–I) Cells were transfected transiently with siGRP78 or control siRNA for 24 hr, and then incubated 4 hours with or without 30 nM MK2206, followed by treatment with 1 μM DOX for 48 hr. Protein levels in the membrane lysates or total lysates of OS cell lines and their resistant sublines were detected by Western blot using GAPDH as loading control. Data are represented as mean ± SD (n = 3).
Figure 5
Figure 5. Knockdown of GRP78 combined with MK2206 inhibits the development of DOX-induced chemoresistance in OS cells in vitro and in vivo
(A) U-2 OS or MG-63 cells were seeded into 12-well culture plates at 0.5 × 105 cells per well overnight. Subsequently, the cells were pretreated with or without 1 mL of complete media containing 5 μg/mL Polybrene for 5-min. The cells were treated with or without shRNA lentiviral particles for GRP78 or control for 24 hr. Then fresh media without Polybrene was placed and cells were treated with 5 nM DOX and/or 30 nM MK2206. When growth of the cells reached 90% confluence, the cells were harvested and reseeded at the density of 0.5 × 105 cells/well, and the DOX concentration increased. Three parallel experiments in each group were performed simultaneously. The experiments were carried out for 245 days (n = 3). (B) The IC50 values of DOX and MK2206 for each cell line shown in Figure 5A. Cells were seeded into 96-well plates at the density of 3500 cells/well. The next day, concentration gradient of chemicals was added and cells were incubated for further 48 hr. MTT assay was performed (n = 3). (C) Protein levels in the membrane lysates or total lysates of OS cells were detected by Western blot with GAPDH as loading control. OS parental sensitive cell lines were used as negative control (n = 3). (D and E) MG-63 cells were infected with the lentiviral constructs. After replacing the fresh medium, cells were diluted to 1:3 for selecting stable clones expressing the shRNA by 5 μg/mL puromycin dihydrochloride. Four weeks later, resistant colonies were picked and expanded for xenograft experiments. Three million MG-63 cells were stably transfected with shGRP78, or control lentiviral particles were suspended in 1:2 PBS/Matrigel in a total volume of 200 μL and subcutaneously injected into the right forelimb of each mouse. When mean tumor volume reached approximately 100 mm3, 30 mice were randomized into five groups (n = 6/group) with approximately equivalent ranges of tumor volume between groups. Mice were treated with MK2206 (60 mg/kg) formulated in 30% Captisol by oral gavage thrice a week. DOX (hydrochloride) (2 mg/kg) was administered by intraperitoneal injection once every 4 days. The control group was treated with 30% Captisol by oral gavage thrice a week. Tumor growth was measured with calipers every other day, and tumor volume was calculated using the following formula: volume (mm3) = length × width2 × 0.5. After 21 days, the animals were sacrificed, and xenografts were removed and weighed (n = 6). Data are represented as mean ± SD.
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