L‐Se‐methylselenocysteine sensitizes lung carcinoma to chemotherapy

Abstract Objectives Organic Selenium (Se) compounds such as L‐Se‐methylselenocysteine (L‐SeMC/SeMC) have been employed as a class of anti‐oxidant to protect normal tissues and organs from chemotherapy‐induced systemic toxicity. However, their comprehensive effects on cancer cell proliferation and tumour progression remain elusive. Materials and Methods CCK‐8 assays were conducted to determine the viabilities of cancer cells after exposure to SeMC, chemotherapeutics or combined treatment. Intracellular reactive oxygen species (ROS) levels and lipid peroxidation levels were assessed via fluorescence staining. The efficacy of free drugs or drug‐loaded hydrogel against tumour growth was evaluated in a xenograft mouse model. Results Among tested cancer cells and normal cells, the A549 lung adenocarcinoma cells showed higher sensitivity to SeMC exposure. In addition, combined treatments with several types of chemotherapeutics induced synergistic lethality. SeMC promoted lipid peroxidation in A549 cells and thereby increased ROS generation. Significantly, the in vivo efficacy of combination therapy was largely potentiated by hydrogel‐mediate drug delivery. Conclusions Our study reveals the selectivity of SeMC in the inhibition of cancer cell proliferation and develops an efficient strategy for local combination therapy.

doses of Se have been found to promote anti-oxidant activity, protecting membrane lipids and macromolecules from peroxidesmediated oxidative damage. [6][7][8] Among the Se compounds, the clinical application of inorganic Se compounds is limited due to their high water solubility, poor liposolubility, and high mutagenic and genotoxic properties. On the contrary, organic Se compounds such as SeMC, selenocystine and selenomethionine pass the cell membrane more efficiently and exhibit fewer side effects and lower systemic toxicity, thus holding great potential in cancer therapy. [9][10][11] Low levels of Se metabolites can incorporate into and form active sites of a number of Se-containing proteins including glutathione peroxidases (GPX), glutathione reductase (GR) and thioredoxin reductases (TrxR), which function as enzymes to regulate intracellular redox status and prevent oxidative damage from exogenous stimuli. [12][13][14][15][16] In contrast, medium-to-high doses of Se compounds lead to increased production of hydrogen selenide (HSe -) and methyl selenol (CH3Se -), which act as pro-oxidants to interfere with intracellular redox balance and induce the formation of superoxide and hydrogen peroxide. 17,18 Consequently, increased ROS generation may render cancer cells more susceptible to chemotherapeutic agents. [19][20][21][22] Therefore, it is reasonable to apply medium-to-high doses of Se compounds, lower the threshold of cancer cells on ROS tolerance and enhance the efficacy of chemotherapy.
Conventional drug administration depends on blood circulation and is apt to cause systemic effect on normal tissues and organs. Besides, the lack of targeted delivery results in low bioavailability of drugs in the tumour tissue with short retention time. 23 In comparison, hydrogel-based drug delivery platform represents an intelligent strategy to address these issues. 24,25 Particularly, in situ formation of hydrogel allows local drug delivery, increases drug concentration at tumour foci and reduces systemic exposure. The design of tumour microenvironment (TME)responsive hydrogel scaffolds enables tunable hydrogel disassembly, providing continuous and controllable release of therapeutic agents. [26][27][28][29][30] Herein, we examined the effects of SeMC on the viabilities of several types of cancer and non-cancer cells and found that the A549 lung adenocarcinoma cell line was more sensitive to SeMC treatment than other cell lines. Combining SeMC with chemotherapeutics such as Epirubicin (EBN), 5-Fluorouracil (5-Fu), Gemcitabine (GEM), Cisplatin (CDDP) or Paclitaxel (PTX) produced synergistic effects on A549 cell death. We further showed that SeMC-induced lipid peroxidation to increase the ROS levels in A549 cells and sensitized them to chemotherapeutic agents. Lastly, we utilized a type of TME-responsive hydrogel for combined local delivery of SeMC and EBN to A549 tumours in a mouse xenograft model and achieved markedly enhanced efficacy on the inhibition of tumour growth comparing to conventional administration.

| Detection of ROS and lipid peroxides
A549 cells (4 × 10 5 cells per well) were cultured in 24-well plates overnight. After incubation with SeMC and/or therapeutic drugs for 12 hours, the cells were washed twice with PBS and incubated with DMEM containing 20 μM DCFH-DA for 45 minutes. Fluorescence imaging was performed on a Leica TCS SP8 confocal laser scanning microscope (Ex = 488 nm, Em = 520 nm). For detection of intracellular lipid peroxides levels, the cells were stained with 2 μM BODIPY C11 in DMEM for 60 minutes after drug treatment and PBS wash and were imaged by the confocal laser scanning microscope (Ex = 500/581 nm, Em = 510/591 nm).

| Animal treatment
BALB/c nude mice (female, 6-7 weeks old) were purchased from SLAC Laboratory Animal Co. Ltd. All mouse experiments were conducted following protocols approved by the Animal Care and Use Committee of Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences.
For tumour cell inoculation, 6 × 10 6 A549 cells stably expressing luciferase were injected subcutaneously in the mice received on the right flank.
When the tumour size reached ~90 mm 3 , the mice were randomly divided into three groups (n = 7 per group) and intratumorally injected with unloaded hydrogel (Gel), free SeMC/EBN (2 mg/kg SeMC and 2 mg/kg EBN) or SeMC/EBN-loaded hydrogel (SeMC/EBN@Gel, 2 mg/kg SeMC and 2 mg/kg EBN) every four days. For hydrogel administration, 100 μL PVA (7.5 wt%) matrix in the absence or presence of drugs was mixed with 100 μL TSPBA (5 wt%) linker in situ. The tumours were measured by a digital calliper and the tutor size was calculated according to the following formula: (length × width 2 )/2. In addition, in vivo bioluminescence imaging was carried out to monitor tutor progression. The mice were anesthetized with isoflurane, injected intraperitoneally with D-Luciferin (150 μg/g) and imaged by an IVIS Spectrum Imaging System (Perkin Elmer).

| Statistical analysis
Statistical analyses were performed with the GraphPad Prism. All data were representative of >3 independent experiments and presented as mean ±SD or mean ±SEM. Student's t test was used to determine the statistical significance of the differences between two groups. ns means not significant, *P < .05, **P < .01, ***P < .001.

| SeMC inhibits A549 cell growth
Firstly, we measured the effect of SeMC on the viability of 293T cells and L6 cells, two representative non-cancer cell lines. At concentrations ranging from 50 to 200 μM, no sign of cytotoxicity was observed ( Figure 1A and Figure S1A). Therefore, we tested several

| SeMC induces lipid peroxidation
Next, we investigated the mechanism under SeMC-induced ROS generation. Although low levels of Se compounds may assist intracellular redox balance, redundant Se exposure can cause adverse effect via the induction of lipid peroxidation. We determined the levels of lipid peroxidation in A549 cells with BODIPY C11, whose fluorescence signal would shift from red to green upon oxidation ( Figure 3A). Individual EBN treatment did not increase the levels of lipid peroxidation in A549 cells. In contrast, incubation with SeMC significantly induced lipid peroxidation, and combined treatment produced an effect similar to that by SeMC alone ( Figure 3B). These data indicated that SeMC disturbed intracellular redox homeostasis independent of therapeutic drugs, providing an alternative route for ROS accumulation.

| Preparation and characterization of ROSresponsive hydrogel
Since both SeMC and EBN could increase intracellular oxidative stress, it would be ideal to restrict their activities mainly in the tumour tissue. Therefore, we resorted to the hydrogel drug delivery  Figure 4D and Figure S4). Collectively, above results confirmed the feasibility of the hydrogel platform as a carrier for spatiotemporally controllable administration of therapeutics.

| Hydrogel-mediated drug delivery enhances anti-tumour activity
Having established the efficiency of drug-loaded hydrogel in cultured cells, we continued to explore its efficacy in mice bearing A549 carcinomas. We intratumorally injected SeMC/EBN@Gel every four days (from day 3 to day 27). As comparison, the control Gel and free SeMC/EBN were injected with the same interval ( Figure 5A).
Bioluminescence signal from the A549 cells indicated that repeated administration with free SeMC/EBN moderately inhibited tumour progression, whereas the SeMC/EBN@Gel dramatically suppressed Meanwhile, we observed no significant changes in the body weight among different groups, suggesting that our therapeutic strategies did not trigger severe side effects ( Figure 5E). Together, these results supported our hypothesis that the in situ formed hydrogel could enhance the anti-tumour efficacy of SeMC and EBN combination therapy.

| D ISCUSS I ON
The comprehensive effects of organic Se compounds on cancer cell proliferation are controversial. [31][32][33] Here, we monitored the viabilities of a series of different cell lines in the presence of SeMC. At concentrations between 50 and 200 μM, most tested cell lines showed good tolerance to SeMC exposure. However, we also found that A549 cells are more susceptible to SeMC treatment than other types of cells including 293T, L6, 4T1, CT26 and Hepa1-6 cells. While these results suggest that the effects of SeMC can be cell-specific, the precise mechanism underlying these differences are not clear. Given SeMC needs to be metabolized before cellular utilization, 34-36 one possible explanation is that different types of cells accumulate harmful metabolites of SeMC at different rates.

The inhibitory effect of SeMC on A549 cells prompted us to
test the efficacy of its combination with chemotherapeutics. We found that SeMC showed a broad spectrum of augmentation on the efficacy of various types of chemotherapeutic agents. Therefore, the combination of SeMC with chemotherapeutics could reduce the required doses of drugs for cancer therapy and avoid potential side effects. We further found that SeMC-induced the generation of lipid peroxides, which might sensitize the A549 cells to chemotherapeutics.
Hydrogel-based drug delivery platforms are promising tools to provide sustained drug release, increase drug concentration at tumour foci and reduce systemic toxicity. 24 Considering the abundant ROS in the TME, 37,38 we constructed a hydrogel ). *P < .05, **P < .01, ***P < .001, ns means not significant scaffold that was responsive to ROS for local SeMC and EBN combination therapy. As expected, treatment with SeMC/EBN@ Gel inhibited tumour progression more efficiently than the free drugs. In our experimental design, SeMC/EBN@Gel was administrated every four days. We expect that the interval of administration can be further prolonged via the improvement of the hydrogel network.
In conclusion, our findings suggest cell-specific effect of SeMC on different types of cancer cells. Our data indicate that SeMC induces lipid peroxidation to increase ROS generation, and thereby potentiates the efficacy of therapeutic agents. We show the potential of SeMC in tumour inhibition via combined treatment with chemotherapeutics and develop a hydrogel-based drug delivery strategy that achieves much higher efficiency than conventional drug administration.

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science

CO N FLI C T O F I NTE R E S T
All authors of this paper declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the findings of this study are available from the corresponding authors upon reasonable request.