Retinoid- and sodium-butyrate-induced decrease in heat shock protein 70 membrane-positive tumor cells is associated with reduced sensitivity to natural killer cell lysis, growth delay, and altered growth morphology

Abstract

Human tumors frequently present heat shock protein 70 (Hsp70) on their cell membranes, whereas corresponding normal tissues fail to do so. Therefore, an Hsp70 membrane-positive phenotype provided a tumor-specific marker. Moreover, membrane-bound Hsp70 provides a target structure for the cytolytic attack mediated by natural killer (NK) cells. Vitamin A derivatives 13-cis retinoic acid (13-RA) and all-trans retinoic acid (ATRA) and sodium-butyrate (SBU) are known for their redifferentiating capacity. Therefore, we asked the question whether loss in tumorigenicity might be associated with a reduced Hsp70 membrane expression. For our studies we used epithelial colon (CX+/CX-) and thyroid (ML-1) cancer cells, with initially different Hsp70 cell surface expression pattern. After treatment up to 7 weeks with freshly prepared 13-RA, ATRA, and SBU at nonlethal concentrations of 10 microM, 1 microM, and 0.5 mM, respectively, growth morphology, Hsp70 levels, and sensitivity toward Hsp70-specific NK cells were compared with that of untreated tumor cells. Significant growth delay was determined in CX+ tumor cells after 6 weeks treatment with 13-RA. Concomitantly, growth morphology changed from spheroid cell clusters to monolayers. Despite a weak increase in cytosolic Hsp70, the percentage of Hsp70 membrane-positive cells dropped significantly after repeated treatments with 13-RA and ATRA in CX+ and ML-1 but not in CX- tumor cells. Similar results were observed with SBU. Functionally, the decrease in Hsp70 membrane-positive CX+ and ML-1 cells correlated with a reduced sensitivity to lysis mediated by NK cells. In summary, redifferentiating agents predominantly affected Hsp70 membrane-positive tumors. The decrease in Hsp70 membrane positivity correlated with a lower sensitivity to NK lysis, growth delay, and altered growth morphology.

Fig 1.

CX+ tumor cell growth was reduced by treatment with 13-cis retinoic acid (13-RA). Adherent growing CX+ and CX− tumor cells were incubated with freshly prepared 13-RA (10 μM) at each individual cell passage (twice a week). After each passage, equal cell counts (0.5 × 10⁶ cells in 5 mL medium) were seeded in T-25 flasks. Absolute counts of viable cells were determined by trypan blue staining after 1 to 6 weeks of treatment in single cell suspensions of adherent and nonadherent cells after trypsinization. The percentage of trypan blue–positive, dead tumor cells before and after treatment was always less than 5%. Growth inhibition data represented mean values of 6 independent experiments ± SD; *marks values significantly different from control (P < 0.05)

Fig 2.

Growth morphology of CX+ tumor sublines changed from cell clusters to monolayers after treatment with 13-cis retinoic acid (13-RA) and all-trans retinoic acid (ATRA). Under physiological conditions, CX+ tumor sublines showed spheroid cell cluster growth (magnification 100×). After treatment with 13-RA (10 μM) for 6 weeks and with ATRA (1 μM) for 5 weeks, cells changed their growth behavior to monolayers. On a single cell level it became obvious that the shape of CX+ tumor cells changed from spheroid to spindle shaped. Scale bar, 200 μm

Fig 3.

13-cis retinoic acid (13-RA) increased cytosolic heat shock protein 70 (Hsp70) levels in CX+ and CX− tumor sublines. Adherent growing CX+ and CX− tumor cells were kept either untreated (ctrl) or were incubated with13-RA (10 μM) at each cell passage. After 6 weeks, cell lysates were prepared and equal protein amounts (10 μg) were run on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Western blots were stained with an Hsp70 and a tubulin-specific antibody, and protein bands were analyzed by densitometry. Increase in cytosolic Hsp70 was related to tubulin. The data represent mean values of 7 independent experiments ± SD. One representative Western blot was shown in the upper part of the graph

Fig 4.

The 13-cis retinoic acid (13-RA) reduced the percentage of Hsp70 membrane-positive cells in the CX+ and ML-1 tumor cells. The percentage of Hsp70 positively stained cells was determined by flow cytometry in untreated (ctrl), 13-RA-treated, all-trans retinoic acid (ATRA)-treated CX+/CX− and ML-1 tumor cells. CX+/CX− tumor cells were treated with 10 μM 13-RA for 4 and 6 weeks (A) and with 1μM ATRA for 5 weeks (B); ML-1 tumor cells were treated with 1 μM 13-RA for 2, 4, and 7 weeks (C). Data represent mean percentages of Hsp70-positively stained cells ± SD of 6 (A,B) and 3 (C) independent experiments; *marks values significantly different from control (P < 0.05)

Fig 5.

13-cis retinoic acid (13-RA) reduced the sensitivity of CX+ and ML-1 tumor cells toward the lytic capacity of NK cells. CX+ (A) and CX− (B) tumor sublines were kept either untreated or were treated with 13-RA (10 μM) for 4 and 6 weeks; ML-1 tumor cells were kept either untreated or were treated with 13-RA (1 μM) for 4 and 7 weeks (C). Untreated and treated tumor cells were used as target cells in a standard 4-hour ⁵¹Cr release assay. CD3-/CD19-depleted natural killer (NK) cells, stimulated for 4 days with Hsp70 peptide TKD (2 μg/ mL) and low dose interleukin-2 (100 IU/mL), were used as effector cells at different effector to target cell (E:T) ratios ranging from 20:1 to 2:1. Purity of CD3-/CD19-negative NK cells was greater 95%. Mean values of 3 independent experiments ± SD were illustrated

Fig 6.

Sodium-butyrate (SBU) reduced the sensitivity of CX+ tumor cells toward the lytic capacity of natural killer (NK) cells. CX+ and CX− tumor sublines were kept either untreated or were treated with SBU (0.5 mM for 48 hours). Untreated and treated tumor cells were used as target cells in a standard 4-hour ⁵¹Cr release assay. CD3-/CD19-depleted NK cells, stimulated for 4 days with heat shock protein 70 (Hsp70) peptide TKD (2 μg/mL) and low dose interleukin-2 (100 IU/mL), were used as effector cells at different effector to target cell (E:T) ratios ranging from 10:1 to 1:1. Purity of CD3-/CD19-negative NK cells was greater 95%. Mean values of 3 independent experiments ± SD were illustrated