Tumor Cell-Derived Microvesicles Induced Not Epithelial-Mesenchymal Transition but Apoptosis in Human Proximal Tubular (HK-2) Cells: Implications for Renal Impairment in Multiple Myeloma

Abstract

Renal impairment (RI) is one of the hallmarks of multiple myeloma (MM) and carries a poor prognosis. Microvesicles (MVs) are membrane vesicles and play an important role in disease progression. Here, we investigated the role of MVs derived from MM cells (MM-MVs) in RI of MM. We found that MM-MVs significantly inhibited viability and induced apoptosis, but not epithelial-mesenchymal transition in human kidney-2 (HK-2), a human renal tubular epithelial cell line. The protein levels of cleaved caspase-3, 8, and 9, and E-cadherin, were increased, but vementin levels were decreased in the HK-2 cells treated with MM-MVs. Through a comparative sequencing and analysis of RNA content between the MVs from RPMI8226 MM cells (RPMI8226-MVs) and K562 leukemia cells, RPMI8226-MVs were enriched with more renal-pathogenic miRNAs, in which the selective miRNAs may participate in the up-regulation of the levels of cleaved caspase-3. Furthermore, the levels of CD138+ circulating MVs (cirMVs) in the peripheral blood were positively correlated with the severity of RI in newly-diagnosed MM. Our study supports MM-MVs representing a previously undescribed factor and playing a potential role in the development of RI of MM patients, and sheds light on the potential application of CD138+ cirMV counts in precise diagnosis of RI in MM and exploring MM-MVs as a therapeutic target.

Keywords: microvesicles; multiple myeloma; renal impairment.

Figure 1

Characterization of myeloma cell-derived microvesicles (MM-MVs) (A,B) Transmission electron microscopy revealing MVs (arrow) as 100–1000 μm vesicles shed from U266 cells (U266-MVs). Scale bar = 2 μm (A) and 0.2 μm (B); (C) Scanning electron microscopy showing typical morphology of MVs derived from RPMI8226 myeloma cells (RPMI8226-MVs). Scale bar = 2 μm; (D,E) Representative flow cytometry analysis of U266-MVs and RPMI8226-MVs revealing the presence of CD138+Calcein-AM+ vesicles, with the 1 μm microbeads for gating the MVs for size verification and Calcein-AM to detect the integrity of MVs.

Figure 1

Characterization of myeloma cell-derived microvesicles (MM-MVs) (A,B) Transmission electron microscopy revealing MVs (arrow) as 100–1000 μm vesicles shed from U266 cells (U266-MVs). Scale bar = 2 μm (A) and 0.2 μm (B); (C) Scanning electron microscopy showing typical morphology of MVs derived from RPMI8226 myeloma cells (RPMI8226-MVs). Scale bar = 2 μm; (D,E) Representative flow cytometry analysis of U266-MVs and RPMI8226-MVs revealing the presence of CD138+Calcein-AM+ vesicles, with the 1 μm microbeads for gating the MVs for size verification and Calcein-AM to detect the integrity of MVs.

Figure 2

Inhibited viability and induced apoptosis in human kidney-2 cells (HK-2 cells) by MM-MVs. (A) HK-2 cells (10⁵/mL) were treated with various concentrations of MM-MVs and K562-MVs (1, 5, 10, and 50 μg/mL) for 24, 48, and 72 h, respectively. The effects of MM-MVs on viability in HK-2 cells were determined with Cell Counting Kit-8 (CCK-8) assay. MM-MVs, but not K562-MVs, inhibited proliferation in HK-2 cells at the time points in a dose-dependent manner; (B) After 48 h, the effects of MM-MVs and K562-MVs on apoptosis in HK-2 cells were determined with FITC-annexin V-PI FCM analysis. MM-MVs, but not K562-MVs, induced apoptosis in HK-2 cells at 48 h in a dose-dependent manner. Representative FCM analysis of early and late apoptosis in HK-2 cells treated with MM-MVs and K562-MVs (10 μg/mL) (top) and bar graphs for apoptosis rates (bottom); (C) The effects of MM-MVs on the morphology of the HK-2 cells treated with MM-MVs or K562-MVs (50 μg/mL) were observed using an charge-coupled Nikon Coolpix 995 digital charge-coupled device (CCD) camera attached to a Nikon Diaphot inverted phase-contrast microscope (Nikon, Tokyo, Japan), Magnification: 100×. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Inhibited viability and induced apoptosis in human kidney-2 cells (HK-2 cells) by MM-MVs. (A) HK-2 cells (10⁵/mL) were treated with various concentrations of MM-MVs and K562-MVs (1, 5, 10, and 50 μg/mL) for 24, 48, and 72 h, respectively. The effects of MM-MVs on viability in HK-2 cells were determined with Cell Counting Kit-8 (CCK-8) assay. MM-MVs, but not K562-MVs, inhibited proliferation in HK-2 cells at the time points in a dose-dependent manner; (B) After 48 h, the effects of MM-MVs and K562-MVs on apoptosis in HK-2 cells were determined with FITC-annexin V-PI FCM analysis. MM-MVs, but not K562-MVs, induced apoptosis in HK-2 cells at 48 h in a dose-dependent manner. Representative FCM analysis of early and late apoptosis in HK-2 cells treated with MM-MVs and K562-MVs (10 μg/mL) (top) and bar graphs for apoptosis rates (bottom); (C) The effects of MM-MVs on the morphology of the HK-2 cells treated with MM-MVs or K562-MVs (50 μg/mL) were observed using an charge-coupled Nikon Coolpix 995 digital charge-coupled device (CCD) camera attached to a Nikon Diaphot inverted phase-contrast microscope (Nikon, Tokyo, Japan), Magnification: 100×. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Activated apoptotic pathways of caspase-3, -8, and -9 and Bcl-2 family members in HK-2 cells treated with MM-MVs. HK-2 cells were treated with MM-MVs and K562-MVs (10 μg/mL) and the expression of proteins was assayed by Western blotting. (A,C) MM-MVs, but not K562-MVs, significantly induced the activation of caspase-3, -8, and -9. Both U266-MVs and RPMI8226-MVs reduced the expression of total caspase-3, -8, and -9, and increased the expression of cleaved caspase-3, -8, and -9; (B,D) MM-MVs up-regulated pro-apoptotic Bim and tBid proteins and down-regulated anti-apoptotic Bcl-xL and Bcl-2 proteins. Bar graphs for (A,B) were shown in (C,D), respectively. Each value was expressed as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control. 2.4. MM-MVs up-regulate E-cadherin protein and down-regulate vimentin protein in HK-2 cells.

Figure 4

MM-MVs up-regulate E-cadherin protein and down-regulate vimentin protein in HK-2 cells. HK-2 cells were treated with MM-MVs and K562-MVs (10 μg/mL) and the expression of proteins was assayed using Western blot and immnofluorescence analysis. MM-MVs, but not K562-MVs, significantly up-regulated E-caderin protein and down-regulated vimentin protein in the HK-2 cells at 48 (A) and 72 h (B). Bar graphs for A and B were shown in (C) and (D), respectively. The two EMT markers were also assayed using a Nikon (Melvile, NY, USA) Eclipse TE300 fluorescence microscope in the HK-2 cells treated with MM-MVs and K562-MVs (10 μg/mL) for 72 h (E) magnification, 200×. Each value was expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 vs. control.

Figure 5

Selective miRNAs in MM-MVs that confer caspase-3-induced apoptosis in HK-2 cells. (A) Comparative analysis of renal-pathogenic miRNAs between RPMI8226-MVs and K562-MVs. RPMI8226-MVs were enriched in more highly-expressed renal-pathogenic miRNAs than did K562-MVs; (B) The heatmap of renal-protective miRNAs in RPMI8226-MVs and K562-MVs showed low levels of renal-protective miRNAs in the two types of MVs; (C,D) A schematic illustration representing MM-MV-packed miRNAs, including renal-pathogenic (C) and –protective miRNAs (D), and potential targets involved in regulation of caspase-3 in HK-2 cells. Yellow triangles: miRNAs relatively highly represented in RPMI8226-MVs. Green ellipse: caspase 3 gene. Blue rectangles: regulators interacted with hub genes and targeted by miRNAs; (E) Evaluation of the levels of the miRNAs involved in the regulation network and differentially expressed between RPMI8226-MVs and K562-MVs, using real-time PCR (* p < 0.01, ** p < 0.05).

Figure 5

Selective miRNAs in MM-MVs that confer caspase-3-induced apoptosis in HK-2 cells. (A) Comparative analysis of renal-pathogenic miRNAs between RPMI8226-MVs and K562-MVs. RPMI8226-MVs were enriched in more highly-expressed renal-pathogenic miRNAs than did K562-MVs; (B) The heatmap of renal-protective miRNAs in RPMI8226-MVs and K562-MVs showed low levels of renal-protective miRNAs in the two types of MVs; (C,D) A schematic illustration representing MM-MV-packed miRNAs, including renal-pathogenic (C) and –protective miRNAs (D), and potential targets involved in regulation of caspase-3 in HK-2 cells. Yellow triangles: miRNAs relatively highly represented in RPMI8226-MVs. Green ellipse: caspase 3 gene. Blue rectangles: regulators interacted with hub genes and targeted by miRNAs; (E) Evaluation of the levels of the miRNAs involved in the regulation network and differentially expressed between RPMI8226-MVs and K562-MVs, using real-time PCR (* p < 0.01, ** p < 0.05).

Figure 6

CD138+ cirMV counts positively correlate with renal impairment. The enrolled 61 de novo patients with MM were divided into two groups according the levels of SCr (group 1, SCr < 2 mg/dL, n = 45; group 2 SCr ≥ 2 mg/dL, n = 16). The levels of CD138+ cirMVs were significantly higher in group 1 than in group 2 (A, p = 0.0138), and the ROC value for using CD138+ cirMV counts to diagnose RI in de novo patients with MM was 0.731 (B).