Ascorbic acid predominantly kills cancer stem cell-like cells in the hepatocellular carcinoma cell line Li-7 and is more effective at low cell density and in small spheroids

doi:10.21203/rs.3.rs-3905688/v1

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Ascorbic acid predominantly kills cancer stem cell-like cells in the hepatocellular carcinoma cell line Li-7 and is more effective at low cell density and in small spheroids

https://doi.org/10.21203/rs.3.rs-3905688/v1

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published 01 Mar, 2024

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The development of therapies that target cancer stem cells (CSCs) is an important challenge in cancer research. The antioxidant system is enhanced in CSCs, which may lead to resistance to existing therapies. Ascorbic acid (AA) has the potential to act as both an antioxidant and a pro-oxidant agent, but its effects on CSCs are a subject of current research. Here, we investigated the effect of AA focusing specifically on CSCs with the hepatocellular carcinoma cell line Li-7. The Li-7 cell line is heterogenous consisting of CD166⁻ and CD166⁺ cells; CD166⁻ cells include CSC-like cells (CD13⁺CD166⁻ cells) and CD13⁻CD166⁻ cells that can revert to CD13⁺CD166⁻ cells. The addition of AA to the culture medium caused cell death in both cell populations in CD166⁻ cells in a concentration dependent manner. In contrast, AA administration had a limited effect on CD166⁺ non-CSC cells. The level of reactive oxygen species after AA treatment was elevated only in CD166⁻ cells. The effect of AA only occurred at low cell densities in 2D and 3D cultures. In a mouse tumor model injected with Li-7 cells, intraperitoneal administration of AA failed to prevent tumor formation but appeared to delay tumor growth. Our findings shed light on why AA administration has not become a mainstream treatment for cancer treatment; however, they also show the possibility that AA can be used in therapies to suppress CSCs.

ascorbic acid

cancer stem cell

cell density

hepatocellular carcinoma

reactive oxygen species

The development of therapies that target cancer stem cells (CSCs) is important in cancer research as these stem cells are responsible for recurrence, metastasis, and resistance to chemotherapy[1–3]. We previously described a hepatocellular carcinoma (HCC) cell line, Li-7, that consists of multiple cell types and contains CSC-like CD13⁺CD166⁻ cells with high tumorigenicity[4]. CD13⁺CD166⁻ cells have the ability to produce both CD13⁻CD166⁻ and CD166⁺ cells; CD13⁻CD166⁻ cells can revert to CD13⁺CD166⁻ cells, but CD166⁺ cells cannot. When CD13⁺CD166⁻ cells are cultured in RPMI1640 supplemented with 10% fetal bovine serum (RPMI) for long periods of time, all cells eventually become CD166⁺ cells and lose their tumorigenicity. We also reported that mTeSR1, a medium for human pluripotent stem cells, maintains CD13⁺CD166⁻ cells in long-term culture[5], and that a set of nine genes is associated with the maintenance of these CSC-like cells[6]. In brief, the Li-7 cell line consists of three cell populations: (1) CD13⁺CD166⁻ cells with high tumorigenicity and CSC characteristics, (2) CD13⁻CD166⁻ cells able to revert to CD13⁺CD166⁻ cells in mTeSR1 culture, and (3) CD166⁺ cells with no tumorigenicity and that cannot revert to CD166⁻ cells.

Ascorbic acid (AA) is a water-soluble vitamin known for its effects on cell growth and differentiation and for its strong antioxidant properties[7, 8]. The antioxidant system is enhanced in CSCs, and can inhibit reactive oxygen species (ROS) accumulation[9]. Although AA has antioxidant properties and can remove ROS, it can also act as a pro-oxidant that generates ROS[10]. The medium mTeSR1 is used for culture of human pluripotent stem cells and can maintain CSC-like cells in the Li-7 cell line; the presence of AA may be responsible for the activation of CSC-like cells through an antioxidant effect. Although cytotoxic effects have been reported for pharmacological doses of AA in various cell lines[10], the effects on HCC and CSCs are not well understood. In this study, we examined the effect of AA on the Li-7 cell line with a particular focus on CSC-like cells.

Cell culture

The human HCC cell line Li-7 (RCB1941) was provided by the RIKEN BRC through the National Bio-Resource Project of MEXT, Japan. CD166⁻ cells were collected by removing CD166⁺ cells using a magnetic beads method. Li-7 cells were cultured with RPMI or mTeSR1 (STEMCELL Technologies) as described in our previous report[5].

Flow cytometric analysis

Li-7 cells cultured with RPMI or mTeSR1 were stained with allophycocyanin-conjugated anti-human CD13 (BD Biosciences) and phycoerythrin-conjugated anti-human CD166 (BD Biosciences). Staining and analytical methods were as described in our previous report[6]. FACS Verse (BD Biosciences) was used for the analyses.

Drug toxicity assay

CD166⁻ and CD166⁺ cells cultured with RPMI were seeded into 96-well plates (5 x 10³ cells/well) and incubated overnight; the medium was then replaced with fresh RPMI or RPMI containing AA (L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate; nacalai tesque), tert-butyl hydroperoxide (TBHP) (Thermo Fisher Scientific), or sulfasalazine (SIGMA). The cells were cultured for 72 hours, then the medium was removed and the cells were washed with phosphate buffered saline (PBS). Cell viabilities were measured using a Cell Counting Kit-8. Absorbance at 450 nm was measured using a 2030 Multilabel Reader (ARVO X3, PerkinElmer). When measuring effects over time, measurements were taken at 0, 24, 48, and 72 hours after the initial change of medium. The effect of cell density on response to treatment was analyzed in cultures containing 0.96–16 x 10³ cells/well.

ROS detection assay

CD166⁻ and CD166⁺ cells cultured with RPMI were seeded into 96-well plates (5 x 10³ cells/well), incubated overnight, and then the medium was replaced with fresh RPMI or RPMI containing 1000 µM of AA. A ROS assay kit (Dojindo) was used to measure intracellular ROS levels at 0, 24, 48, and 72 hours after changing the medium; fluorescence intensity at excitation 485 nm/ emission 535 nm was measured using a 2030 Multilabel Reader.

Spheroid formation assay

CD13⁺CD166⁻ cells cultured in mTeSR1 were seeded into a 96-well NanoCulture plate-MS (ORGANOGENIX) (0.5 or 1 x 10⁴ cells/well) with 150 µL of RPMI. Fifty µL of medium was replaced with fresh medium twice a week. The number of spheres with a diameter greater than 50, 100, and 150 µm was counted on day 15 using a microscope equipped with a digital camera (DP25, Olympus). AA (1000 µM) was added between days 1 to 5 to the cultures.

Tumor formation in mice

Five-week-old female BALB/c nu/nu nude mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). CD13⁺CD166⁻ cells (5x10⁶ cells) cultured with mTeSR1 were suspended in 200 µL of RPMI and injected subcutaneously in the left and right sides of the back of the mice. AA treatment was initiated when the tumor diameter was larger than 5 mm. Either 200 µL saline or 20 mg AA in 200 µL saline were administered intraperitoneally 4 times a week. Tumor volume was measured weekly and calculated as [length x (width)² x 0.5]. All recipient mice were sacrificed at 6 weeks after injection, and volume and weight of tumors were measured and calculated.

Statistics

Student’s t-test was performed to identify statistically significant differences. A value of P < 0.05 was considered significant.

Pharmacological doses of AA kill CD166⁻ cells in cultures of Li-7 cells

To examine the effect of AA on Li-7 cells cultured with RPMI, AA was added at various doses. The addition of AA selectively killed CD166⁻ cells in a dose-dependent manner (Fig. 1A). In contrast, the addition of AA had no effect on any cells in cultures using mTeSR1 (Fig. 1B). CD166⁻ cells that had been cultured in mTeSR1 died when the medium was replaced with RPMI containing AA (Fig. 1C). These results showed that the cytotoxicity of AA is restricted to cells in cultures using RPMI. Almost all CD166⁻ cells in RPMI cultures were dead at 72 hours after the addition of AA (Fig. 1D); no effect of AA was observed on CD166⁺ cells (Fig. 1E). Thus, CD166⁻ cells but not CD166⁺ cells are sensitive to AA in a dose-dependent manner (Fig. 1F).

Increase in ROS levels after AA treatment

It is well known that pharmacological doses of AA cause increases in ROS[10, 11]. We therefore investigated whether the levels of ROS were increased by the addition of AA. After the addition of AA, significantly increased ROS levels were observed in CD166⁻ cells after 24 hours (Fig. 2A) but no increase was observed in CD166⁺ cell cultures (Fig. 2B). To determine the effects of ROS on CD166⁻ and CD166⁺ cells, TBHP, which generates ROS, was added to the culture medium. TBHP induced death of CD166⁻ cells at a lower concentration than for CD166⁺ cells (Fig. 2C). In addition, treatment with sulfasalazine, which inhibits the uptake of cystine required for synthesis of glutathione to decrease ROS, showed a similar result (Fig. 2D). These findings suggest that the addition of AA increases ROS in CD166⁻ cells and that this increase was responsible, at least in part, for the increased rate of cell death.

Effect of AA varies with cell density and spheroid size

To examine the effects of AA in the culture condition closest to tumor tissue, we added AA to spheroid cultures. AA was added from day 1 to day 5 after the start of culture. We found that the number and the size of spheres were reduced by addition of AA and this effect was largest when AA was added earlier (Fig. 3A, 3B). Next, we added AA (1000 µM) to cell cultures of different densities. Cell viability remained constant at higher cell densities but fell significantly at lower cell densities (Fig. 3C, 3D). A somewhat similar result was obtained with the spheroid formation assay: the reduction in number and size of spheroids was more apparent in cultures with smaller spheroids (Fig. 3F) than in those with larger spheroids (Fig. 3E). These results indicate that AA is more effective at lower cell densities and on smaller spheroids.

AA can inhibit tumor growth in mouse

AA was intraperitoneally injected into nude mouse with subcutaneous tumors. Tumor growth in the treated group was significantly reduced compared to the control group (Fig. 4A, 4B). Tumor volume and weight were also significantly reduced in the AA treated group at 6 weeks after transplantation (Fig. 4C, 4D). Although AA did not prevent tumor formation, it did inhibit tumor formation to some extent.

Although the effects of AA exposure on cancer cells have been demonstrated in in vitro culture[10], two in vivo randomized controlled trials[12, 13] failed to identify any action of oral AA administration against cancer. However, as intravenous administration of AA could increase the blood concentration of AA [14] then it is still possible that AA may have a role in cancer therapy. The potential anticancer mechanisms of AA include ROS generation, prevention of HIF-1 activation, and epigenetic regulation via the ten-eleven translocation protein. AA is also expected to promote anti-programmed death ligand 1 (PD-L1) therapy[15]. In the present study, we show that AA induced an increase in ROS only in CD166⁻ cells cultured in RPMI; we also showed an increased rate of CD166⁻ cell death as ROS levels increased. Although AA did not induce cytotoxic effects in cells cultured in mTeSR1, we did note that if AA was added to cells cultured in mTeSR1 supplemented with FBS, cytotoxicity was observed, although it was weaker than the effect seen in RPMI cultures (supplemental Fig. 1). It is possible that factors in FBS are required for AA cytotoxicity and/or that mTeSR1 contains factors that inhibit ROS. We also found that the cytotoxic effect of AA was restricted to cultures with low cell densities, and even in these AA did not result in total cell death. Extrapolating from these findings, it seems that AA monotherapy for advanced cancer with large tumors is likely to have limited efficacy; this may be one of the reasons for the failure of AA monotherapy clinical trials[16, 17]. In order to achieve an effect using AA therapy for advanced cancers, it may be necessary to combine AA with other drugs that target non-CSCs or that have been shown to enhance antitumor effects, such as anti-PD-L1 therapy[18].

CSCs have an crucial role in cancer recurrence[1] and are an important therapeutic target. The efficacy of AA against CSCs has been described previously [19], and a clear cytotoxic effect on CSC-like cells in the Li-7 cell line was also obtained in this study. However, a small proportion of CD166⁻ cells can survive high concentrations of AA and TBHP, and these cells may be the most important therapeutic targets to avoid cancer recurrence. We also found that AA is highly effective against small spheroids including CSCs and it has been shown to suppress metastasis in mouse xenograft model[20]. Since AA is well tolerated in terms of side effects, AA monotherapy or combined with adjuvant chemotherapy may have considerable potential for suppressing postoperative recurrence.

In this study, we examined the effects of AA treatment on the Li-7 cell line that contains CSC-like cells. AA predominantly killed CSC-like cells following the generation of ROS; the effect of AA was more clearly observed in cultures at lower cell densities and on smaller spheroids. These results suggest that AA may have the potential for use in the clinic as an approach to suppress CSCs, although further work is clearly required to determine the optimal treatment.

Acknowledgments

We thank all the members of Cell Engineering Division of RIKEN BioResource Research Center for secretarial and technical assistances.

Funding Information

This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT).

Conflict of Interest

The authors have no conflict of interest.

Ethics approval

Research protocol was approved by Institutional Reviewer Board. All animal experiments were approved by the Institutional Animal Care and Use Committee of RIKEN Tsukuba Branch.

Author Contributions:

Methodology, Y.S and K.S; experiment, Y.S and K.S.; analysis, Y.S; writing – original draft, Y.S.; writing – review & editing, K.S. and Y.S; supervision, T.Y., K.T., K.S, and Y.N.

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Download PDF

Journal Publication

published 01 Mar, 2024

Read the published version in Biochemical and Biophysical Research Communications →

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You are reading this latest preprint version

Ascorbic acid predominantly kills cancer stem cell-like cells in the hepatocellular carcinoma cell line Li-7 and is more effective at low cell density and in small spheroids

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Cell culture

Flow cytometric analysis

Drug toxicity assay

ROS detection assay

Spheroid formation assay

Tumor formation in mice

Statistics

Results

Pharmacological doses of AA kill CD166⁻ cells in cultures of Li-7 cells

Increase in ROS levels after AA treatment

Effect of AA varies with cell density and spheroid size

AA can inhibit tumor growth in mouse

Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

Ascorbic acid predominantly kills cancer stem cell-like cells in the hepatocellular carcinoma cell line Li-7 and is more effective at low cell density and in small spheroids

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Cell culture

Flow cytometric analysis

Drug toxicity assay

ROS detection assay

Spheroid formation assay

Tumor formation in mice

Statistics

Results

Pharmacological doses of AA kill CD166− cells in cultures of Li-7 cells

Increase in ROS levels after AA treatment

Effect of AA varies with cell density and spheroid size

AA can inhibit tumor growth in mouse

Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

Pharmacological doses of AA kill CD166⁻ cells in cultures of Li-7 cells