3.1 Tetrandrine Citrate reduced the viability and induced the cell death in LUAD cells

Figure 1 A described the internal chemical molecular structure of Tetrandrine. As Tetrandrine is a hydrophobic alkaloid with low solubility in water, Tetrandrine Citrate was formed by mixing Tetrandrine free base with citric acid at 4:1 ratio in ddH2O. TetC has a solubility of up to 500 mg/ml in water. Next, in order to investigate the anticancer properties of TetC on LUAD, different doses of TetC were treated in the LUAD cells (A549 and H1299). We found that TetC had the potentiality to restrain the viability of LUAD cells in a concentration-dependent manner (Fig. 1B, C). And the half-maximal inhibitory concentrations (IC50s) in A549 and H1299 cells were 10.610 µM and 9.492 µM, respectively, therefore, 10 µM of TetC concentration were finally selected for the following experiments. In addition, as shown in Fig. 1D–F, TetC obviously prevented the growth of A549 and H1299 cells in a concentration-dependent manner. Furthermore, we utilized the Annexin V-FITC/PI assay to evaluate the effect of TetC on the LUAD cell death. As expected, treatment with TetC increased the amounts of A549 and H1299 cells death compared with the ctrl-group (Fig. 1G–I). Collectively, these data suggested that TetC inhibited the viability and induced cell death in LUAD cells.

Fig. 1

TetC reduced the viability and induced the cell death in LUAD cells. A Chemical structure of Tet. B CCK-8 assay depicted TetC inhibited A549 cell in a concentration- (0–30 µM) and time- (12 h, 24 h, and 48 h) dependent manner. C CCK8 assay depicted TetC inhibited H1299 cell in a concentration- (0–30 µM) and time- (12 h, 24 h, and 48 h) dependent manner. D–F Morphologic features of A549 and H1299 cells on microscopy. Cells became obviously round and showed shrunk after TetC (5 µM and 10 µM) treatment for 24 h. G A549 and H1299 cells were subjected to TetC (5 µM and 10 µM) for 24 h and followed by Annexin V-FITC/PI assay. H, I Quantification of Annexin V-FITC/PI in A549 and H1299 cells. *p < 0.05; **p < 0.01; ***p < 0.001

3.2 Tetrandrine citrate induced DNA damage and inhibited the clonogenesis of LUAD cells

Immunofluorescence was used to measure the change of γ-H2AX expression which is a marker for early DNA damage. Results showed that TetC dramatically increased amounts of γ-H2AX foci in a dose-dependent manner of A549 and H1299 cells (Fig. 2A–D). Next, the clonogenic assay was employed to identify the effect of TetC on cell clone. The results depicted in Fig. 2E–G showed that compared with the control group, TetC remarkably inhibited the colony formation of LUAD cells in a dose-dependent manner. In summary, our research has shown that TetC plays a significant role in inducing DNA damage and suppressing the clonogenesis of LUAD cells, highlighting its potential as a valuable tool in cancer treatment and prevention.

Fig. 2

TetC induced DNA damage and inhibited the clonogenesis of LUAD cells. A A549 cell were treated with TetC (5 µM and 10 µM) for 24 h and stained with γ-H2AX antibody. DAPI stands for nucleus staining. Scale bar: 10 μm. B Quantification of γ-H2AX immunofluorescence in A549 cell. C H1299 cell were treated with TetC (5 µM and 10 µM) for 24 h and stained with γ-H2AX antibody. DAPI stands for nucleus staining. Scale bar: 10 μm. D Quantification of γ-H2AX immunofluorescence in H1299 cell. E The colony formation assay of A549 and H1299 cells were performed under treatment of TetC (5 µM and 10 µM) for 14 days. F, G Quantification of colony formation in A549 and H1299 cells. *p < 0.05; **p < 0.01; ***p < 0.001

3.3 Ferroptosis as an important method contributed to tetrandrine citrate-induced LUAD cell death

Several studies conducted in the past year have demonstrated that stimulation of ferroptosis plays an irreplaceable function in the chemoresistance of various human cancers [20, 21]. In this work, we observed that TetC-induced LUAD cell death was remarkably blocked by three different ferroptosis-related inhibitors, ferrostatin-1 (Fer-1, 5 μm, ferroptosis inhibitor), liproxstatin-1 (Lip-1, 2 μm, ferroptosis inhibitor) and defer-oxamine (DFO, 20 μm, iron chelator), proving that ferroptosis might be essential for TetC-induced cell death in LUAD (Fig. 3A–F). We then looked into the morphology of the TetC treated cells. As shown the results of transmission electron microscopy (TEM) in Fig. 3G, the treated cells played shrinking mitochondria, condensed mitochondrial membrane densities and diminished or disappeared mitochondria cristae, which are emblematic morphological features of ferroptosis.

Fig. 3

TetC induced ferroptosis in LUAD cells. A, D The viability of A549 and H1299 cells was detected following TetC (5 µM and 10 µM) with or without Fer-1 (5 µM) treatment for 24 h. B, E The viability of A549 and H1299 cells was detected following TetC (5 µM and 10 µM) with or without DFO (20 µM) treatment for 24 h. C, F The viability of A549 and H1299 cells was detected following TetC (5 µM and 10 µM) with or without Lip-1 (2 µM) treatment for 24 h. *p < 0.05; **p < 0.01; ***p < 0.001. G The ultrastructure of control and TetC (10 µM) treated LUAD cell was observed using TEM. TEM: transmission electronic microscopy

To investigate whether TetC induced LUAD cell death through ferroptosis, we examined ferroptosis-related marker proteins (Ferritin heavy chain, FTH; Prostaglandin endoperoxide synthase 2, PTGS2; Divalent metal transporter 1, DMT1) by western blot. Our data reveled that LUAD cells treated with TetC displayed a dose-dependent increase in the expression of PTGS2 and DMTI, but decrease the levels of FTH (Fig. 4A–C). Furthermore, the addition of Fer-1 could reverse the expression of these proteins induced by TetC treatment (Fig. 4D–K).

Fig. 4

Ferroptosis was triggered by TetC in LUAD cells. A The expression of ferroptosis-related proteins in A549 and H1299 cells were detected after TetC (5 µM and 10 µM) treatment for 24 h by western blotting. B Quantification of western blotting in A549 cell. C Quantification of western blotting in H1299 cell. D The expression of ferroptosis-related proteins in A549 cell were detected after TetC (10 µM) treatment with or without Fer-1 (5 µM) for 24 h by western blotting. E–G Quantification of western blotting in A549 cell. H The expression of ferroptosis-related proteins in H1299 cell were detected after TetC (10 µM) treatment with or without Fer-1 (5 µM) for 24 h by western blotting. I–K Quantification of western blotting in H1299 cell. *p < 0.05; **p < 0.01; ***p < 0.001

As part of the ferroptosis process, it is essential to note the depletion of GSH, as well as the accumulation of MDA and ROS [22]. Thus, the level of GSH, MDA and ROS were measured and observed. As expected, the gradually decreased GSH level and the increased MDA level were observed as TetC concentration (5 and 10 μm) over 24 h (Fig. 5A–D). Moreover, Fer-1 could eliminate these results induced by TetC (Fig. 5E–H). As anticipated, the TetC treatment (10 µM, 24 h) upregulated the levels of ROS, and Fer-1 could eliminate this phenomenon in LUAD cells (Fig. 5I–K). In short, these results reveled that ferroptosis acted as the critical mechanism by which TetC triggered cell death in LUAD.

Fig. 5

Ferroptosis contributed to TetC-induced cell death in LUAD. A, B GSH-PX and MDA levels in A549 cell were detected after TetC (5 µM and 10 µM) treatment for 24 h. C, D GSH-PX and MDA levels in H1299 cell were detected after TetC (5 µM and 10 µM) treatment for 24 h. E, F GSH-PX and MDA levels in A549 cell were detected after TetC (10 µM) treatment with or without Fer-1 (5 µM) for 24 h. G, H GSH-PX and MDA levels in H1299 cell were detected after TetC (10 µM) treatment with or without Fer-1 (5 µM) for 24 h. I–K ROS level in A549 and H1299 cells was detected after TetC (10 µM) treatment with or without Fer-1 (5 µM) for 24 h with a ROS Assay Kit. *p < 0.05; **p < 0.01; ***p < 0.001

3.4 Tetrandrine citrate induced ferroptosis in LUAD cells via SLC7A11/GPX4 axis

To investigate the potential molecular mechanisms of TetC-induced ferroptosis, we selected two pivotal signals from the ferroptosis complicated pathway, SLC7A11 and GPX4, as the focus of our next research. The expression of SLC7A11 and GPX4 were examined by western blot, reveling that they were remarkably decreased following TetC treatment, and could rescue by the addition of Fer-1 in LUAD cells (Fig. 6A–J). Immunofluorescence assay further found that not only the expression level of SLC7A11 and GPX4 proteins were significantly reduced, but also the fluorescence intensity of these two proteins was weakened with TetC treatment, which could be reversed by Fer-1 (Fig. 6K, L). We further transfected SLC7A11 with overexpressing plasmid and the si-RNA targeting SLC7A11 in LUAD cells before TetC treatment to confirm whether TetC-induced ferroptosis occurred through SLC7A11. As indicated in Fig. 7A–E, K, L, SLC7A11 upregulation clearly increased the decline in GPX4 protein level and GSH content triggered by TetC (10 µM, 24 h). Meanwhile, Fig. 7F–J proved that si-SLC7A11 reduced GPX4 protein levels induced by TetC in A549 and H1299 cells. In addition, the overexpression of SLC7A11 remarkably attenuated the TetC-induced (10 µM, 24 h) accumulation of MDA and ROS (Fig. 7M–Q). Taken together, TetC induced ferroptosis in LUAD through regulating the SLC7A11/GPX4 axis.

Fig. 6

The expression of SLC7A11 and GPX4 in LUAD cells after TetC treatment. A SLC7A11 and GPX4 levels in A549 and H1299 cells were detected after TetC (5 µM and 10 µM) treatment for 24 h by western blotting. B–E Quantification of western blotting in A549 and H1299 cells. F SLC7A11 and GPX4 levels in A549 and H1299 cells were detected after TetC (10 µM) treatment with or without Fer-1 (5 µM) by western blotting. G–J Quantification of western blotting in A549 and H1299 cells. K The fluorescence intensity of A549 cell was detected after TetC (10 µM) treatment with or without Fer-1 (5 µM) by Immunofluorescence staining. L The fluorescence intensity of H1299 cell was detected after TetC (10 µM) treatment with or without Fer-1 (5 µM) by Immunofluorescence staining. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 7

TetC-induced ferroptosis depends on the SLC7A11/GPX4 signaling pathway. A SLC7A11 and GPX4 levels in A549 and H1299 cells were detected after TetC (10 µM) treatment with or without oe-SLC7A11 by western blotting. B-E  Quantification of western blotting in A549 and H1299 cells. F SLC7A11 and GPX4 levels in A549 and H1299 cells were detected after TetC (10 µM) treatment with or without si-SLC7A11 by western blotting. G–J Quantification of western blotting in A549 and H1299 cells. K, L GSH-PX and MDA levels in A549 cell were detected after TetC (10 µM) treatment with or without oe-SLC7A11. M, N GSH-PX and MDA levels in H1299 cell were detected after TetC (10 µM) treatment with or without oe-SLC7A11. O–Q ROS level was detected after TetC (10 µM) treatment with or without oe-SLC7A11 with ROS Assay Kit. *p < 0.05; **p < 0.01; ***p < 0.001

3.5 Tetrandrine citrate benefited to treating LUAD and triggered ferropotosis in vivo

To evaluate the potential antitumor efficacy of TetC in vivo, the amount of A549 (2 × 10 7) cell was expertly subcutaneously injected into the armpits of experimental BALB/c nude mice (4 weeks age). Once the mice developed 80–100 mm3 tumors, they were randomly assigned to receive intraperitoneal injections of TetC (100 mg/kg/day), TetC (100 mg/kg/day) + Fer-1 (50 mg/kg/day) or the same volume of saline for approximately 3 weeks (Fig. 8A). Our data showed that TetC dramatically inhibited tumour growth and Fer-1 could abolished this effect (Fig. 8B, C). Furthermore, we detected the effects of TetC on ferroptosis through RT-qPCR, which also reduced SLC7A11, GPX4 and FTH and increased PTGS2 following the TetC treatment. Moreover, the cell death induced by TetC was apparent eliminated by co-treatment with Fer-1 (Fig. 8D–G). Finally, immunohistochemical (IHC) were performed on tumor sections to further detect protein expression. As demonstrated in the results, The decreased levels of SLC7A11, GPX4 and Ki67 were found following the TetC treatment, which could be reversed by Fer-1 (Fig. 8H). In summary, the above data demonstrated that TetC had the efficacy for inhibiting tumour growth and partly induced ferroptosis in LUAD in vivo.

Fig. 8

TetC benefited to treating lung cancer in vivo. A Once the tumours reached 80–100 mm3, the model mice received three different treatments (saline, 100 mg/kg/day TetC, 100 mg/kg/day TetC + 50 mg/kg/day Fer-1) per day until the experiment terminated. B The photograph of tumor samples in three different treatment groups. C Tumor size was measured. D–G RT-qPCR analysis of SLC7A11, GPX4, PTGS2 and FTH mRNA in tumors from three different treatment groups. H The SLC7A11, GPX4 and Ki67 were determined by IHC. *p < 0.05; **p < 0.01; ***p < 0.001

3.6 The potential biosafety value of tetrandrine citrate on tumour treatment

In further research, we looked forward to exploring whether TetC had a good safety profile for tumor suppression. We collected serum from the TetC-treated mice to examine the function of their liver and kidneys. Our founding indicated that there were no significant changes in serum ALT and AST levels after TetC intervention (Fig. 9A, B). The CRE and BUN results also testified the lack of highlighted renal dysfunction in the three drug-treated mice (Fig. 9C, D). The complete heart, kidney, and liver tissues were dissected and extracted from the mice in each group, and performed HE staining after a series of preliminary treatments. TetC treatment did not trigger significant intrinsic toxic reactions in mice of each group (Fig. 9E). Altogether, our results suggested that TetC had a degree of biosafety in LUAD treatment through the vivo experiment.

Fig. 9

The potential biosafety value of Tetrandrine citrate on tumor treatment. A, B The levels of ALT and AST were determined from the serum of mice after three different treatment groups (saline, 100 mg/kg/day TetC, 100 mg/kg/day TetC + 50 mg/kg/day Fer-1). C, D The levels of CRE and BUN were determined from the serum of mice after three different treatment groups (saline, 100 mg/kg/day TetC, 100 mg/kg/day TetC + 50 mg/kg/day Fer-1). E HE staining of heart, kidney and liver tissues in mice. ALT and AST: markers of liver function; CRE and BUN: markers of renal function. *p < 0.05; **p < 0.01; ***p < 0.001