The role of ROS in CAB- and SKF83959-induced cell death. (A, B) Western blotting showing expression of SOD1, but not SOD2, was decreased after SKF83959 treatment in vitro or in vivo. SOD activity was also reduced upon SKF83959 treatment. (C, D) N-acetylcysteine (NAC), a ROS inhibitor, reversed the CAB- or SKF83959-induced increase of ROS level, from 2.7-fold to 1.6-fold (p
Figure 5.
Activation of DRD5 induces autophagy…
Figure 5.
Activation of DRD5 induces autophagy and blocks autophagic flux in pituitary tumor cells.…
Figure 5. Activation of DRD5 induces autophagy and blocks autophagic flux in pituitary tumor cells. (A) MMQ cells were treated with the DRD2 agonist quinpirole at 100 μM (left panel), the DRD5 agonist SKF83959 at 25 μM (right panel), or the DRD5 antagonist SKF83566 at 50 μM (middle panel). Western blotting showed that only treatment with the DRD5 agonist SKF83959 resulted in an increase in the LC3-II level. (B) The DRD5 agonist SKF83959 (12.5 μM)-induced LC3-II increase was partially abolished by the DRD5 antagonist SKF83566. (C) TEM showed the formation of autophagosomes in MMQ cells treated with the DRD5 agonist SKF83959 (SKF83959 vs. Ctrl: 7.2 ± 1.3 vs. 0.9 ± 0.2, n = 40, p < 0.01) but not with the DRD2 agonist quinpirole or the DRD5 antagonist SKF83566. (D) The DRD5 agonist SKF83959 increased LC3 puncta in GH3 cells, indicating the activation of autophagy. ***, p < 0.001. (E-F) Western blotting showed increased SQSTM1 protein expression upon SKF83959 treatment (E), but there was no major change at the Sqstm1 mRNA level by qRT-PCR (F). (G) Flow analysis showing the curve of pH value shifting to the right, indicating that intracellular pH was decreased by SKF83959. (H) Fluorescence microscopy showed the lysosomal proteolytic cleavage. DQ-Red-BSA exhibited bright red fluorescence at 24 h, but not at 48 h and 72 h upon SKF83959 treatment in GH3 cells. (I) Western blotting showing the expression of CTSD and CTSB after SKF83959 treatment of GH3 cells. The expression of mature CTSD and mature CTSB were decreased following the increase of SKF83959 concentration or by SKF83959 (25μM) over time.
Figure 6.
Critical role of autophagy in…
Figure 6.
Critical role of autophagy in DRD5-mediated suppression of pituitary tumor cell growth. (A)…
Figure 6. Critical role of autophagy in DRD5-mediated suppression of pituitary tumor cell growth. (A) Knockdown (KD) of ATG5, ATG7, and BECN1 protein by RNAi in GH3 cells. (B) Quantification of ATG5, ATG7, and BECN1 protein KD in GH3 cells. (C) Viable cell counts in GH3 cells with Atg5, Atg7, or Becn1 KD treated with the DRD5 agonist SKF83959, showing the abolishment of SKF83959-induced growth suppression by Atg5, Atg7, or Becn1 KD. (D) Immunoblots analysis of c-CASP3, PARP and LC3 in GH3 cells after Atg7 KD. (E) 3-MA (5 mM) partially abolished and chloroquine (20 μM) increased DRD5 agonist SKF83959-induced growth suppression in GH3 cells. (F) 3-MA (5 mM) partially abolished and chloroquine (20 μM) increased DRD5 agonist SKF83959-induced LC3-II and SQSTM1 accumulation. NC, nonspecific negative control siRNA; **, p < 0.01; ***, p < 0.001.
Figure 7.
Growth suppression by DRD5 activation…
Figure 7.
Growth suppression by DRD5 activation in human pituitary tumor primary cultures. (A) Western…
Figure 7. Growth suppression by DRD5 activation in human pituitary tumor primary cultures. (A) Western blotting showing DRD5 expression in human pituitary adenomas of different types. NP, normal human pituitary; PRL, PRL-secreting pituitary adenoma; PRL+GH, human pituitary adenoma secreting both PRL and GH; GH, GH-secreting pituitary adenoma; ACTH, ACTH-secreting pituitary adenoma; NFPA, clinically non-functioning pituitary adenoma. (B) Primary cultures of 5 human pituitary adenomas were treated with DRD5 agonist SKF83959 at 25 μM. The viable cell counts were measured at the indicated time points. SKF83959 decreased viable cells significantly in 4 of 5 pituitary tumor primary cultures. (C) Immunostaining showed high levels of DRD5 in 4 pituitary adenomas responsive to SKF83959 but a very low level of DRD5 in one nonresponsive pituitary tumor.
Figure 8.
Growth suppression and autophagy activation…
Figure 8.
Growth suppression and autophagy activation by DRD5 activation in human cancer cells. (A)…
Figure 8. Growth suppression and autophagy activation by DRD5 activation in human cancer cells. (A) SKF83959 (25 μM) treatment at 48 h significantly reduced the viable cell count in human glioblastoma cell lines U87, U251, SHG66, colon cancer cell line SW480, and gastric cancer SCG7901, as measured by MTS assays. (B) Western blotting showing that in these human cancer cell lines, SKF83959 treatment (25 μM) for 48 h resulted in a decrease of p-MTOR and p-EIF4EBP1 and an increase in LC3-II, c-CASP3 and c-PARP, indicating inhibition of MTOR signaling and activation of autophagy. (C) SKF83959 increased LC3-associated green fluorescence in these human cancer cell lines, indicating the activation of autophagy. (D) TEM showed the formation of autolysosomes in SCG7901 and SHG66 cells. (E) DRD5 agonist SKF83959 suppressed tumor growth from human gastric cancer SCG7901 in nude mice. (F) Comparison of SCG7901 tumors from mice treated with control vehicle and with SKF83959 (1 mg/kg) at d 11 of drug administration. (G) Comparison of the ratio of tumor:mouse weights from mice injected with SCG7901 treated with control vehicle and with SKF83959 at d 11 of drug administration. (H) Western blotting showed that compared with the tumors from the control mice the tumors from SKF83959-treated mice expressed more LC3-II, c-CASP3, c-PARP, and less p-MTOR and p-EIF4EBP1. *, p
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