A Pharmacological Strategy Using Stemofoline for more Efficacious Chemotherapeutic Treatments Against Human Multidrug Resistant Leukemic Cells

Abstract

Our previous study reported that stemofoline (STF) exhibited a synergistic effect with chemotherapeutic drugs in human multidrug-resistant (MDR) leukemic cells (K526/Adr) by inhibiting the function of P-glycoprotein, which is a membrane transporter that is overexpressed in several types of MDR cancers. This study further investigated the effects of a combination treatment of STF and doxorubicin (DOX) in vitro and in vivo. The combination treatment of 50 mg/kg of STF strongly enhanced the anti-tumor activity of DOX in SCID-beige mice bearing K562/Adr xenografts without additional toxicity when compared to the single treatment groups. Additionally, an examination of the proliferation markers (Ki67) and the apoptotic marker (TUNEL) in tumor tissues in each group revealed that the combination therapy significantly reduced Ki67 positive cells and increased apoptotic cells. From the in vitro experiments we also found that this combination treatment dramatically induced G1 and G2M arrest in K562/Adr when compared to a single treatment of DOX. STF treatment alone did not show any cytotoxic effect to the cells. These results suggest that the accumulation of DOX enhanced by STF was sufficient to induce cell cycle arrest in K562/Adr. These findings support our previous in vitro data and indicate the possibility of developing STF as an adjuvant therapy in cancer treatments.

Keywords: Chemotherapy; P-glycoprotein; Xenograft model; Stemofoline.

Figure 1

Structure of STF

Figure 2

Potentiation of Antitumor Effects of DOX by STF in K562/Adr Xenograft Model. Mice were treated with saline (control); STF alone at 50 mg/kg; DOX alone at 2 mg/kg; doxorubicin at 2 mg/kg plus STF 50 mg/kg (STF was given 30 minutes before DOX administration). Tumor volumes were monitored during the experiment by a Vernier caliper (D0-D16) (A). After scarification (day 16) time, tumors were removed for measure tumor volume (B) and tumor weight (C). Changes in body weight during the experiment were recorded (D). Microphotographs of H&E sections of internal organs from SCID-Beige mice after treatment with saline (control); STF alone at 50 mg/kg; DOX alone at 2 mg/kg; doxorubicin at 2 mg/kg plus STF 50 mg/kg (STF was given 30 minutes before DOX administration). Mice were sacrificed for pathologic examination 4 day after the last administration. Original magnification (x20) (E)

Figure 3

Tumor Masses were Cross-Sectioned and Fixed in 10% Formalin Buffer and Prepared in Paraffin Embed Block. They were then stained with H and E and immunohistochemistry (A); (Ki67) proliferation marker and apoptotic marker (TUNEL). % of proliferation marker (Ki67)-positive cells (B) and % of apoptotic marker (TUNEL)-positive cells (C) were analyzed using Image J software.

Figure 4

MDR Modulation Effect of STF on DOX Sensitivity in K562/Adr (A) and K562 Cell Lines (B) was determined. The co-administration of DOX (0-40 µM) with or without STF was performed by MTT colorimetric assay for 48 hrs. The mean value from three independent experiments is shown and error bars indicating SD (n = 3) are shown as ***p <0.001 versus DOX single administration at each indicated concentration.

Figure 5

STF Enhanced Sensitivity of K562/Adr Cells to DOX. The graphs represent living cells (A), early apoptosis (B) and late apoptosis cell populations (C). K562/Adr cells were treated with various concentrations of DOX (0-5 µM) with or without 5 µM of STF for 48 hrs. After treatment, K562/Adr were washed twice with PBS and stained with annexin V and propidium iodide (PI). Apoptosis induction was determined by flow cytometry. The mean values from three independent experiments are shown and error bars indicate SD (n = 3).

Figure 6

STF Enhanced Sensitivity of K562/Adr Cells to DOX. The graphs represent G1 (A), S (B) and G2/M phase distribution (C). K562/Adr cells were treated with various concentrations of DOX (0-5 µM) with or without 5 µM of STF for 48 hrs. After treatment, K562/Adr cells were washed twice with PBS and stained with propidium iodide (PI). Cell cycle phase distribution was determined by flow cytometry. The mean value from three independent experiments is shown and error bars indicate SD (n = 3). *p <0.05, **p <0.01 and ***p <0.001 versus DOX single administration at each indicated concentration.