**1. Introduction**

Hepatocellular carcinoma (HCC) is the second most common cause of cancer mortality worldwide [1]. Since HCC is highly aggressive and metastatic, only about 10% of patients have limited options, such as surgical resection, liver transplantation and local ablation [2]. HCC is also one of the most common chemotherapy-resistant tumors. The continuous administration of conventional chemotherapeutic agents and antitumor immune agents causes side effects, such as tumor resistance and poor prognosis. Therefore, it is imperative

**Citation:** Wang, K.; Wang, X.; Zhang, M.; Ying, Z.; Zhu, Z.; Tam, K.Y.; Li, C.; Zhou, G.; Gao, F.; Zeng, M.; et al. Trichosanthin Promotes Anti-Tumor Immunity through Mediating Chemokines and Granzyme B Secretion in Hepatocellular Carcinoma. *Int. J. Mol. Sci.* **2023**, *24*, 1416. https://doi.org/10.3390/ ijms24021416

Academic Editors: Barbara De Filippis, Marialuigia Fantacuzzi and Alessandra Ammazzalorso

Received: 30 November 2022 Revised: 4 January 2023 Accepted: 10 January 2023 Published: 11 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

to explore new drugs or therapeutic strategies targeting HCC [3]. Researchers have identified numerous plant-derived extracts with potent antitumor properties, and representative ones include paclitaxel [4], curcumin [5], millipedium [6], Trichosanthin [7], etc.

Trichosanthin (TCS), a single-chain ribosome-inactivating protein extracted from the tuberous root of the traditional Chinese herb *Trichosanthes*, exhibits prospective application in clinical drug abortion, anti-virus, anti-tumor and immune regulation [8]. Numerous studies have shown that TCS could directly inhibit the proliferation and apoptosis of cancer cells by regulating the expression of Bcl-2 [9], inducing S-phase cell cycle arrest in cancer cells [10], inhibiting tumor dysplasia-related signaling pathways [11], increasing the expression or activation of caspase family proteins [12–14], etc. In physiological conditions, TCS could regulate the immune status of the body by regulating the CD4+/CD8+ T-cell ratio and producing related immune cytokines in peripheral blood [15]. Studies have shown that TCS could enhance the sensitivity of tumors to chemotherapeutic drug treatment [13,16]. Recombinant TCS has shown potent anti-tumor effects [17–19]. It is crucial to investigate the anti-tumor mechanisms and potential applications of TCS.

Granzyme B (GrzB) is an extremely high anti-tumor bioactive protein produced mainly by CD8+ T cells and NK cells [20]. Numerous studies have shown that GrzB can rapidly activate caspase 3-related signaling pathways in target cells [21], which in turn promote cancer cell apoptosis or inflammatory death [22]. Our previous works on immunodeficient nude mice found that the combination of TCS and GrzB had a positive effect in inhibiting HCC, and TCS enhanced the translocation of GrzB from mannose-6-phosphate receptors (M6PR) to HCC cells [23]. However, whether TCS could inhibit HCC by regulating antitumor immunity has not been examined yet. In this study, we used TCS to treat HCC cells and a xenograpft tumor model to investigate the mechanism of TCS regulating the recruitment of T cells in the host immune response against HCC.

#### **2. Results**

#### *2.1. TCS Reduces the Viability of HCC Cells in Culture*

To test if TCS was able to inhibit HCC cell growth in culture, TCS (concentration ranged from 1.5625 to 400 μg/mL) was administered to the H22 HCC cell line for 24, 48 and 72 h. TCS inhibited H22 cell viability in a dose-dependent manner (Figure 1A). The IC50 of HCC cells treated with TCS for 48 h and 72 h was approximately 25 μg/mL (Figure 1A). Then, we treated HCC cells with 25 μg/mL TCS and assayed cell viability at multiple time points, and we observed a significant decrease of cell viability after 36 h (Figure 1B). The Calcein-AM/PI assay also showed a significant and time-dependent increase of dead HCC cells after TCS treatment (Figure 1C). PARP, a nucleus polymerase that appears to be involved in DNA repair and that is a common apoptosis marker cleaved by Caspase-3 [24], was also induced by TCS in a dose-dependent manner (Figure 1F). To further confirm TCS might impair cell viability, the apoptosis inhibitor Z-VAD-FMK was applied to treat HCC cells for 48 h, combined with TCS at multiple concentrations. Z-VAD-FMK significantly inhibited the cell death and PARP cleavage induced by TCS (Figure 1E,F). This suggested that TCS triggered HCC cell death mainly by promoting caspase activities.

#### *2.2. TCS Promoted HCC Cell Death via Apoptosis*

Apoptosis and autophagy are common modes of tumor cell death [25,26]. It has been reported that TCS can promote the death of Oral squamous cell carcinoma SCC25 by inducing cell apoptosis [9]. In order to better understand the mechanisms of TCS-induced cell death, HCC cells were treated with 25 μg/mL TCS, total protein was extracted and the expression levels of apoptosis- and autophagy-related proteins were detected by Western blot. Apoptosis usually involves the activation of a series of caspase enzymes. The upstream caspase of the intrinsic pathway is caspase 9, while the exogenous pathway is caspase 8; after that, internal and external pathways converge to caspase 3 [27]. Western blot analysis showed that Caspase 9, Caspase 8 and Caspase 3 were all decreased in HCC cells after 72 h

of TCS treatment (Figure 2A). Instead, the level of Cleaved-caspase 9, Cleaved-caspase 8 and Cleaved-caspase 3 were all elevated after 48 h of TCS treatment (Figure 2A).

**Figure 1.** Effects of TCS on the cell viability and death of HCC cells. (**A**) H22 HCC cells were treated with different doses of TCS for 24 h, 48 h and 72 h. CCK-8 assay with absorbance at 450 nm was used

to evaluate the cell viability; (**B**) IC50 dose of TCS (25 μg/mL) was used to treat H22 HCC cells for 12 h, 24 h, 36 h, 48 h, 60 h and 72 h. CCK-8 assay with absorbance at 450 nm was used to evaluate the cell viability; (**C**) 25 ug/mL TCS treated HCC cell lines at different times with the ratio of dead cells to live cells; (**D**) IC50 dose of TCS (25 μg/mL) was used to treat H22 HCC cells for 0 h, 24 h, 36 h and 72 h. The Calcein-AM/PI method was used to detect dead or alive cells, with green as live cells and red as dead cells. Bar = 100 μm; (**E**) H22 HCC cells were treated with different doses (0, 12.5, 25 and 50 μg/mL) of TCS. Meanwhile, 40 μM caspase inhibitor (Z-VAD-FMK) was used in combination. Calcein-AM/PI assay was used to detect dead or alive cells; green is live cells, red is dead cells. Bar = 100 μm; (**F**) Western blot assay for PARP and Cleaved-PARP protein expression after 48 h of TCS and Z-VAD-FMK coadministration. \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001.

**Figure 2.** Effects of TCS on apoptosis and autophagy of HCC cells. (**A**) H22 HCC cells were treated with 25 μg/mL TCS for 24 h, 48 h and 72 h. Western blot assayed the levels of key apoptosis proteins Caspase 9, Cleaved-caspase 9, Caspase 8, Cleaved-caspase 8, Caspase 3 and Cleaved-caspase 3; (**B**) H22 HCC cells were treated with 25 μg/mL TCS for 12 h, 24 h, 36 h and 48 h. Western blot assayed the levels of key autophagy proteins P62 and LC3A/B. NS means no significant difference, \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001.

Studies have also shown that TCS can inhibit the growth of gastric cancer cell MKN-45 by inducing autophagy [28]. In the process of autophagosome formation, LC3I is lipidized to form LC3II; therefore, LC3I/LC3-II is considered a marker of autophagosome. In addition, the autophagic receptor p62 is also commonly used as an autophagic marker [29]. However, our results show that the level of the autophagy markers P62 and LC3I/LC3II were not significantly different between the control and TCS-treated groups (Figure 2B). This indicated that TCS did not induce significant autophagy in HCC cells. Therefore, TCS induced HCC cell death, mainly via apoptosis.
