Shikonin Inhibits Cancer Through P21 Upregulation and Apoptosis Induction

Abstract

Shikonin is a natural naphthoquinone compound and has demonstrated potent anti-cancer activities; however, the underlying molecular mechanisms remained elusive. Here we report that Shikonin inhibited the growth of a wide range of human cancer cell lines, illustrating a broad anticancer effect. Mechanistically, we show that Shikonin arrested the cell cycle at the G2/M phase, inhibited the ERK-dependent cell growth signal, and induced cell death in both P53 wild type and mutant cancer cells, which collectively contributed to the growth inhibitory effect of Shikonin. A pan-apoptosis inhibitor largely suppressed Shikonin-induced cell death, suggesting an important role of apoptosis in this process. Intriguingly, Shikonin also activated autophagy and inhibition of autophagy by depleting critical autophagic genes further increased Shikonin-induced cell death, indicating a protective role of autophagy. In uncovering the molecular mechanisms underlying these effects of Shikonin, we found that Shikonin induced a robust upregulation of P21 independent of the P53 status, upregulated autophagy genes, as well as inhibited expression of genes required for cell growth. Using mouse tumor models, we confirmed the strong anticancer effect of Shikonin in vivo. Together, our data reveal a broad range of pharmacological functions of Shikonin, involving simultaneous growth inhibition, cell cycle arrest, autophagy activation and apoptosis induction through regulating expression of critical genes involved in these pathways. Our study may facilitate the development of Shikonin in cancer therapy as a single agent or in combination with other anticancer therapies.

Keywords: P21; Shikonin; anticancer effect; apoptosis; autophagy; cell cycle arrest.

Figure 1

Chemical structure of Shikonin.

Figure 2

Effects of Shikonin on cancer cell growth. A549 (A), PANC-1 (B), U2OS (C), MDA-MB-231 (D), or LO2 (E) cells were treated with indicated concentrations of Shikonin for 48 h, and cell survival was analyzed by the CCK-8 assay. Data were normalized to that of DMSO control and presented as relative survival rate from five replicates. IC₅₀ values were analyzed using the GraphPad software. (E) A549 cells were treated with DMSO or different concentrations of Shikonin or 10 μM doxorubicin (DOX) for 1, 2, 3 and 4 days, and cell survival was analyzed by the CCK-8 assay. The absorbance of Shikonin-treated group was normalized to that of the same day DMSO control, which yields the relative survival rate. Data represent mean and standard deviation from five replicates. (F) A549 cells were treated with DMSO or different concentrations of Shikonin for 1, 2, 3 and 4 days, and cell survival was analyzed by CCK-8 assay. The absorbance of Shikonin-treated group was normalized to that from the same day DMSO control, which yields the relative survival rate. Data represent mean and standard deviation from five replicates.

Figure 3

Effects of Shikonin on cell survival and cell cycle. (A) A549 or PANC-1 cells were treated with increasing concentrations of Shikonin for 24 h, and clonogenic survival assay was measured. Representative images are shown. (B) Quantitation of relative survival from (A). Data represent mean and standard deviation from five replicates. *P <0.001 compared with the DMSO control. (C) MDA-MB-231, U2OS or LO2 cells were treated with 5 μM of Shikonin for 24 h, and clonogenic survival assay was measured. Representative images are shown. (D) A549 or PANC-1 cells were treated with 5 μM of Shikonin for 0, 12 and 24 h, fixed and cell cycle profile was analyzed. (E) Quantitation of cell cycle distribution for A549 and PANC-1 cells from D. Data represent average and standard deviation from two independent experiments.

Figure 4

Shikonin induces cell death. (A) PANC-1 cells were treated with 5 μM of Shikonin for 0, 12 and 24 h, and protein expression was measured using specific antibodies. I and II indicate non-modified and lipidated forms of LC3B, respectively. Anti-PARP recognizes both full-length and cleaved PARP proteins, and the latter is indicated by the arrow. (B) Quantitation of dead cell population from cells in (A) by trypan blue staining. Data represent mean and standard deviation from five replicates. *P <0.001. (C) MDA-MD-231 cells were treated with 5 μM of Shikonin for 0, 12 and 24 h, and protein expression was measured by specific antibodies. (D) Quantitation of dead cell population from C by trypan blue staining. Data represent mean and standard deviation from five replicates. *P <0.001. SW620 (E) or HT-29 (F) cancer cells were treated with 5 μM of Shikonin for 12 h, and protein expression was measured by specific antibodies. (G) Quantitation of dead cell population from E and F by trypan blue staining. Data represent mean and standard deviation from five replicates. *P < 0.001.

Figure 5

Apoptosis-dependent cell death induction by Shikonin. (A) A549 cells were treated with 5 μM of Shikonin for 0, 12 and 24 h, and apoptotic cells were analyzed by Annexin V staining. (B) PANC-1 cells were treated with 5 μM of Shikonin in the presence or absence of 10 μM Z-VAD for 24 h, and protein expression was measured by specific antibodies. Cleaved PARP, LC3B forms I and II are indicated by arrows. (C) Quantitation of dead cell population from B by trypan blue staining. Data represent mean and standard deviation from five replicates. *P < 0.001.

Figure 6

Shikonin activated autophagy. (A) A549 control or ULK1 depleted cells were treated with 5 μM of Shikonin for 0, 12 and 24 h, and protein expression was measured. (B) Quantitation of dead cell population from (A) by trypan blue staining. Data represent mean and standard deviation from five replicates. (C) HeLa control or BECN1 stably depleted cells were treated with 5 μM of Shikonin for 0, 12 and 24 h, and protein expression was measured using specific antibodies. (D) Quantitation of dead cell population from C by trypan blue staining. Data represent mean and standard deviation from five replicates. *P < 0.05.

Figure 7

Shikonin upregulates P21 and alters gene transcription. (A) A549, (B) MDA-MB-231, (C) PANC-1 and (D) U2OS cells were treated with 5 μM of Shikonin for 6 h, and protein expression was measured by specific antibodies. For qPCR analysis, A549 cells were treated with 5 μM Shikonin for 0, 4, 8, 12 and 24 h, RNA was isolated, and expression of genes including CDKN1A (E), TP53 (F), c-FOS (G), JUN (H), ATG5 (I), and ATG3 (J) was analyzed. Data represent mean and standard deviation from five replicates. *P < 0.001 between 0 and 24 h groups.

Figure 8

Mouse studies of Shikonin. (A) 5–6 week old nude mice (n = 2) were injected with vehicle control or 1, 3, 5, 10 or 20 mg/kg Shikonin i.p. twice a week for 2 weeks, and mouse survival was plotted by the Kaplen–Mier plotter. (B) Body weight of mice in (A) was recorded and plotted over time. (C) For nude mice tumor study, 5 * 10⁶ A549 cells were inoculated into nude mice subcutaneously. When the tumor reached around 100 mm³ in size, the mice were treated with 2 mg/kg of Shikonin i.p. twice a week for 3 weeks. Tumor volumes were measured during this time period. Data represent mean and standard error of tumor volume from six mice per group. *P < 0.001.