**LJP-A-3-5-3 Fraction**

**LJP-1~12** RP-HPLC

**Scheme 1.** Key Steps in the isolation of LJPs (LJP-1~12).

#### *4.3. Cell Apoptosis and Cell Cycle Assays*

Ab MTT assay was used to detect the cytotoxicity activity of LJPs in vitro [42]. Briefly, 5 × 10<sup>3</sup> cells/well were seeded in 96-well plates, and varying concentrations of LJP were added. After 48 h, the MTT solution (5 mg/mL) was added for an additional 4 h. The absorbance was measured at 570 nm using a microplate reader (Bio-Rad; Hercules, CA, USA) after 100 μL of DMSO was added. The IC<sup>50</sup> values were determined by the non-linear multipurpose curve-fitting program.

After treatment with LJP-1 (0, 0.36, and 1.80 mM representing control, low dose of LJP-1 and LJP-1-L, and high dose of LJP-1 and LJP-1-H, respectively) for 48 h, the apoptosis of H22 cells was evaluated by flow cytometry. Briefly, after LJP-1 treatment, H22 cells were collected, washed with cold PBS, suspended in binding buffer (100 μL) (BD Biosciences, San Jose, CA, USA), treated with annexin V and propidium iodide (PI) (BD Biosciences), and incubated in the dark for 15 min. Then, another 300 μL binding buffer was added, and flow cytometry analysis was performed for 1 h to measure the rate of apoptosis. The percentages of cells in the G0/G1, S, and G2/M phases were determined using a cell cycle detection kit (BD Biosciences, Haryana, India) using a Beckman Coulter EPICS ALTRA II cytometer (Beckman Coulter, Brea, CA, USA).

#### *4.4. Western Blot Analysis*

After treatment with LJP-1 (0, 0.36, and 1.80 mM representing control, low dose of LJP-1 and LJP-1-L, and high dose of LJP-1 and LJP-1-H, respectively) for 48 h, H22 cells were collected, and protein was collected and measured with a Bradford protein assay to calculate the amount of protein. Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a polyvinylidene difluoride (PVDF) membrane. Immunoblots were blocked with 5% non-fat milk and incubated with primary antibody (1:1000) at 4 ◦C overnight, followed by incubation with the second conjugated peroxidase antibody (1:5000) at room temperature for 2 h [43]. Protein bands were measured, and *β*-actin was used as an internal standard of process control. The blot band densitometry was analyzed with ImageJ software, version 1.53t.

#### *4.5. Immunization of Liver-Cancer-bearing Mice*

The procedures for the animal study were approved by the Animal Ethics Committee of Shenzhen University. Mice were kept in a specific pathogen-free facility with free access to food and water. H22 cells were cultured and suspended in PBS, and 18 Balb/c mice were subcutaneously injected with H22 cells (1 × 106) in the right back near the skin of the underarm. Tumor growth was measured every 2 days. When tumor volume reached 300 mm3, mice were randomly divided into three groups and treated with the following procedures: Group 1 (Control): mice were injected with 200 μL saline every 4 days around tumor tissue; Group 2 (LJP-1-L): mice were injected with LJP-1 (in 200 μL saline) 2 mg/kg every 4 days (4 days in a cycle, four times) around tumor tissue; Group 3 (LJP-1-H): mice were injected with LJP-1 (in 200 μL saline) 10 mg/kg every 4 days (4 days in a cycle, four times) around tumor tissue. All mice were sacrificed on day 23. The subcutaneous tumors were excised, and the tumor index was calculated. Furthermore, tumor tissues were cut into small woven pieces and fixed with polyformaldehyde, and paraffin sections were made later.

#### *4.6. Evaluation of Tumor Apoptosis and Hematoxylin and Eosin (H&E) Staining*

Apoptotic cells in cancer tissue sections were stained with a TUNEL reagen<sup>t</sup> kit following the manufacturer's instructions. Hematoxylin and eosin (H&E) staining was performed according to the routine procedure. The paraffin section was dewaxed and dehydrated with xylene and gradient alcohol, then stained with hematoxylin solution for 5 min, soaked in 1% acid ethanol (1% HCL in 70% ethanol) for 2 min, and rinsed in distilled water. Slices were stained with eosin solution for 3 min, dehydrated with graded alcohol, and cleaned with xylene.

## *4.7. Statistical Analysis*

Data are presented as mean ± standard deviation (SD). GraphPad Prism 5.0 (Graph Pad Software, La Jolla, CA, USA) was used for statistical analysis. Statistical analysis was performed with one-way analysis of variance (ANOVA). *p* < 0.05 was considered statistically significant.

## **5. Conclusions**

In conclusion, our study demonstrates that LJP-1 isolated from *L. japonica* possesses strong antitumor effects against HCC-based on evidence from both in vitro and in vivo studies. In vivo experiments show that LJP-1 strongly suppressed tumor growth on day 14 after treatment at doses of 2 and10 mg/kg. Our data supports that LJP-1 controls liver cancer proliferation by inducing HCC cell apoptosis through the caspase-dependent pathway and by arresting cells in the G0/G1 phase by regulating cell cycle checkpoint proteins. Furthermore, LJP-1 induces caspase-dependent apoptosis, in part, by inhibiting MAPK signaling pathways. The PI3K/AKT pathway and possibly other pathways may be involved. In addition, our screening study shows that other LJPs, namely LJP-4, LJP-8, LJP-9, LJP-11, and LJP-12, also exhibit antitumor activities. Therefore, *L. japonica peptides* (not just LJP-1) could be potential drug candidates for the novel treatment of human liver cancer and warrant further investigation.

**Author Contributions:** Conceptualization, Y.L. (Yiguang Lin) and H.C.; methodology, software, and validation, Y.W., Y.L. (Yuanhui Li), W.G., J.L., W.L. and P.H.; formal analysis, investigation, and data curation, Y.W., Y.L. (Yuanhui Li), W.G. and J.L.; writing—original draft preparation, Y.L. (Yiguang Lin), J.L. and Y.W.; writing—review and editing, Y.W., Y.L. (Yuanhui Li), W.G., J.L., W.L., P.H., Y.L. (Yiguang Lin) and H.C.; visualization, Y.W., J.L., Y.L. (Yiguang Lin) and H.C.; supervision, H.C. and Y.L. (Yiguang Lin); project administration, Y.L. (Yiguang Lin) and H.C.; funding acquisition, H.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The procedures for the animal study were approved by the Animal Ethics Committee of Shenzhen University (No: 2017-0113).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data (figures and tables) used to support the findings of this study are included within the article.

**Conflicts of Interest:** The authors declare no conflict of interest.
