Midkine Mediates Intercellular Crosstalk between Drug-Resistant and Drug-Sensitive Neuroblastoma Cells In Vitro and In Vivo

Abstract

Resistance to cytotoxic agents has long been known to be a major limitation in the treatment of human cancers. Although many mechanisms of drug resistance have been identified, chemotherapies targeting known mechanisms have failed to lead to effective reversal of drug resistance, suggesting that alternative mechanisms remain undiscovered. Previous work identified midkine (MK) as a novel putative survival molecule responsible for cytoprotective signaling between drug-resistant and drug-sensitive neuroblastoma, osteosarcoma and breast carcinoma cells in vitro. In the present study, we provide further in vitro and in vivo studies supporting the role of MK in neuroblastoma cytoprotection. MK overexpressing wild type neuroblastoma cells exhibit a cytoprotective effect on wild type cells when grown in a co-culture system, similar to that seen with doxorubicin resistant cells. siRNA knockdown of MK expression in doxorubicin resistant neuroblastoma and osteosarcoma cells ameliorates this protective effect. Overexpression of MK in wild type neuroblastoma cells leads to acquired drug resistance to doxorubicin and to the related drug etoposide. Mouse studies injecting various ratios of doxorubicin resistant or MK transfected cells with GFP transfected wild type cells confirm this cytoprotective effect in vivo. These findings provide additional evidence for the existence of intercellular cytoprotective signals mediated by MK which contribute to chemotherapy resistance in neuroblastoma.

Figure 1

Co-culture effect on cellular response to doxorubicin. Wild type human neuroblastoma (SK-N-SH WT) cells co-cultured with SK-N-SH WT cells (WT/WT) were compared to co-cultures of SK-N-SH WT cells with doxorubicin resistant cells (SK-N-SH DoxR) (WT/DoxR). Co-cultures were treated with and without doxorubicin at 10⁻⁷ or 10⁻⁶M for 48 hours. Surviving cells were quantified using trypan blue staining. The cell survival ratio represents the number of live WT cells in the drug-treated co-culture divided by the number live cells in the untreated (control), *P < 0.001.

Figure 2

Effect of midkine overexpression on wild type cell survival. A midkine overexpressing SK-N-SH cell line (SK-N-SH HMK) was created as previously described [6]. (a) Midkine expression in the medium of wild type (WT), doxorubicin resistant (DoxR), and HMK cells were analyzed using western blot. SK-N-SH WT, DoxR, and HMK were cultured in 25 cm flasks to 80% confluence. Medium was then exchanged for 2 mL of low fetal bovine serum culture medium and cultured for an additional 48 hours. Cultured medium was then collected and frozen at −80°C prior to being subjected to western blot. The volume of medium loaded on the gel was normalized based on cell count in the flask prior to medium collection. Cell membranes were isolated through a well-established protocol. We have previously demonstrated that midkine transfected cells (HMK) have acquired some doxorubicin resistance [6]. The MTT cell survival assay was performed to determine if SK-H-SH HMK cells have acquired resistance to other chemotherapeutic drugs including (b) etoposide and (c) cisplatin. (d) SK-N-SH WT, DoxR, and HMK cells were treated with or without doxorubicin at 10⁻⁷M for 24 hours prior to collection of cell lysates. Proteins were extracted and probed using western blot for Pgp and β-actin.

Figure 3

Effect of midkine overexpression on cellular response to doxorubicin in co-culture conditions. (a) GFP-transfected wildtype SK-N-SH cells (GFP-WT) were co-cultured with wild type (WT), doxorubicin resistant (DoxR), or human midkine overexpressing SK-N-SH cells (HMK), incubated with (+) or without (−) doxorubicin at 10⁻⁷M for 48 hours. Photographs were taken using fluorescence microscopy. (b) The viability percentage of fluorescent cells (GFP-WT) after each treatment was determined through Hoechst 33342 staining and cell counting. Data represent an average of three independent determinations +/− SE.

Figure 4

Effect of midkine siRNA on cytoprotection. (a) ELISA assay and (b) western blot were used to confirm decreased expression of midkine in doxorubicin resistant SK-N-SH cells (DoxR) treated with midkine. Culture medium from SK-N-SH wild type (WT), DoxR, DoxR cells treated with scramble sequence siRNA (DoxR-scramble), and DoxR cells treated with siRNA to midkine (DoxR-si-midkine) was harvested after growth for 96 hours. (c) SK-N-SH WT cells were grown in co-culture with WT, DoxR, and DoxR-scramble or DoxR-si-midkine cells. Co-cultures were incubated for 48 hours with or without doxorubicin at 10⁻⁷M and 10⁻⁶M. SK-N-SH WT cell survival was then quantified through cell counting after staining with trypan blue. Data represents the average of 4 experiments +/− SE. (d) OSA WT cells were grown in co-culture with OSA WT, DoxR, and DoxR-scramble or DoxR-si-midkine cells. Co-cultures were incubated for 48 hours with or without doxorubicin at 10⁻⁷M and 10⁻⁶M. OSA WT cell survival was then quantified through cell counting after staining with trypan blue. Data represents the average of 4 experiments +/− SE.

Figure 5

Effect of midkine siRNA on doxorubicin resistant cellular response to doxorubicin. siRNA was used to knock down midkine expression in doxorubicin resistant SK-N-SH cells (DoxR) to determine if loss of midkine expression results in restoration of drug sensitivity in the DoxR cells. Wild type (WT), DoxR, DoxR cells treated with scramble sequence RNA (DoxR-scramble) and SK-N-SH DoxR cells treated with siRNA to midkine (DoxR-si-midkine) were cultured with or without doxorubicin at 10⁻⁷M and 10⁻⁶M for 24 hours. Cell survival was assayed using trypan blue and cell counting. Data represents the average of 4 experiments +/− SE.

Figure 6

Effect of midkine cytoprotection on growth of wild type human neuroblastoma tumors in vivo. Mice were injected with drug-sensitive human neuroblastoma cells (SK-N-SH GFP-WT) and (a) drug-resistant human neuroblastoma cells (SK-N-SH DoxR) or (b) midkine transfected cells (SK-N-SH HMK) at various ratios (GFP-WT : DoxR or GFP-WT : HMK). Once the tumors were palpable, the mice received injections of doxorubicin (2.5 mg/kg) intraperitoneally every 3 days for a total of 3 doses. Tumor volumes were measured every 3 days for up to 3 weeks. Tumor growth curves in the different groups were generated. Each data point in the growth curve represents the mean of 7 determinations ± SE. *P < 0.05; **P < 0.01; ***P < 0.001. (c and d) Apoptotic GFP-WT cells were measured by TUNEL assay in tumor sections. The histogram represents a summary data.

Figure 7

Immunohistochemistry for midkine in human neuroblastoma tissue samples. Prechemotherapy and postchemotherapy patient neuroblastoma biopsy samples were collected and stained for midkine. Midkine staining was scored using a 0 (negative), 1+ (weak staining), 2+ (moderate staining), and 3+ (strong staining) scale on a blinded basis. Midkine staining was then correlated with tumor stage, N-MYC amplification status, histology (favorable or unfavorable), and survival. Biopsy scores were also evaluated for a change in midkine staining prechemotherapy to postchemotherapy. Shown here is a sample neuroblastoma histology slide stained for midkine.