Skip to main content
NCBI home page
Cancer Immunology, Immunotherapy : CII logoLink to Cancer Immunology, Immunotherapy : CII
. 2005 Jul 23;55(2):166–177. doi: 10.1007/s00262-005-0016-7

Vesiculated alpha-tocopheryl succinate enhances the anti-tumor effect of dendritic cell vaccines

Lalitha V Ramanathapuram 1, Tobias Hahn 1, Michael W Graner 2, Emmanuel Katsanis 2, Emmanuel T Akporiaye 1,✉
PMCID: PMC11029922  PMID: 16041582

Abstract

Alpha tocopheryl succinate (α-TOS) is a non-toxic vitamin E analog under study for its anti-cancer properties. In an earlier study, we showed that α-TOS, when used in combination with non-matured dendritic cells (nmDC) to treat pre-established tumors, acts as an effective adjuvant. In this study, we have used vesiculated α-TOS (Vα-TOS), a more soluble form of α-TOS that is relevant for clinical use, in combination with dendritic cells to treat pre-established murine tumors. We demonstrate that Vα-TOS kills tumor cells in vitro and inhibits the growth of pre-established murine lung carcinoma (3LLD122) as effectively as α-TOS. The combination of Vα-TOS plus non-matured or TNF-α-matured DC is more effective at inhibiting the growth of established tumors than Vα-TOS alone. We also observed that Vα-TOS induces expression of heat shock proteins in tumor cells and that co-incubation of non-matured DC with lysate derived from Vα-TOS-treated tumor cells leads to DC maturation evidenced by up-regulation of co-stimulatory molecules and secretion of IL-12p70. This study therefore demonstrates the immunomodulatory properties of Vα-TOS that may account for its adjuvant effect when combined with DC vaccines to treat established tumors.

Keywords: Dendritic cell, α-tocopheryl succinate, Immunotherapy, Vaccine, Heat shock proteins

Introduction

Alpha-tocopheryl succinate (α-TOS) is an esterified redox inactive analog of vitamin E [27]. Numerous studies have shown that α-TOS is selectively toxic to tumor cells while showing minimal toxicity towards normal cells [15, 26, 28, 35, 36]. In addition to inducing apoptosis of various murine and human cancer cell lines in vitro [11, 15, 22, 29, 35, 36], α-TOS has been shown to inhibit in vivo growth of several experimental tumors including melanoma, breast, lung and colon cancers [5, 23, 24].

Although the in vivo results have been promising, the translation of α-TOS therapy to the clinic is hindered by the lipophilic nature of α-TOS and hence its insolubility in aqueous solvents. In the reported animal studies, α-TOS was dissolved in either ethanol, dimethylsulfoxide or sesame oil [23, 24, 29, 33] all of which are impractical for use in humans. Vesiculated α-TOS (Vα-TOS) is a novel formulation of the drug that arises spontaneously in the presence of sodium hydroxide and sonication [19]. It is readily soluble in aqueous solutions and has been shown to inhibit the progression of tumors as well as prolong the survival of tumor-bearing mice [19].

In an earlier study using the 3LL murine lung tumor model, we showed that the anti-tumor activity of α-TOS plus DC immunotherapy was superior to that of α-TOS alone. We also demonstrated a positive correlation between tumor growth retardation and IFN-γ production by T lymphocytes isolated from the spleens of mice treated with the combination of α-TOS plus DC [29].

In this study, we investigated the effect of Vα-TOS on 3LL Lewis lung tumor cells and its role as an adjuvant of DC-based immunotherapy of established murine lung tumors. We show that Vα-TOS is as tumoricidal as α-TOS in vitro and inhibits the in vivo growth of pre-established murine lung tumors. Vα-TOS used in combination with non-antigen pulsed and non-matured or TNF-α-matured DC inhibited the growth of pre-established tumors more efficiently than Vα-TOS alone. We also observed that co-incubation of soluble components of Vα-TOS-treated tumor cells with non-matured DC causes up-regulation of the co-stimulatory molecules, CD40, CD80 and CD86 and increases the production of IL-12p70. In trying to understand the mechanism by which Vα-TOS causes DC maturation, we showed that Vα-TOS induces the expression of tumor heat shock proteins (hsp60, 70 and 90), which are known to provide “danger” signals that lead to DC maturation [7, 9, 13, 32, 37]. In addition, blocking of the cognate hsp receptor CD91 on nmDC inhibited the up-regulation of the maturation markers CD40, CD80 and CD86 when DC were co-incubated with supernatant derived from tumor cells exposed to Vα-TOS. These findings suggest that tumor growth suppression by Vα-TOS is likely due to its combined effects of tumor cell killing and activation of dendritic cells.

Materials and methods

Chemicals and reagents

Alpha-tocopheryl succinate (α-TOS), Alpha tocopherol (α-TOH) and Alpha-2 macroglobulin (α2M) were purchased from Sigma Chemical Co. (St. Louis, MO). Murine IL-4, GM-CSF and TNF-α were purchased from Peprotech (Rocky Hill, NJ). The antibodies for phenotyping DC (anti-CD11c, anti-I-Ab, anti-CD40, anti-CD80, anti-CD86) were purchased from BD Pharmingen (San Diego, CA) and Caltag Laboratories (Burlingame, CA). The hsp-specific antibodies (hsp60, 70 and 90) were purchased from Stressgen Biotechnologies (Victoria, BC, Canada). The ALEXA Flour 488 antibody was purchased from Molecular Probes (Eugene, OR). The goat anti-mouse HRP-conjugated antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Annexin-V FLOUS staining kit was purchased from Roche Applied Sciences (Indianapolis, IN). The mouse IL-12p70 ELISA kit was purchased from Pierce Biotechnologies (Rockford, IL).

Preparation of vesiculated α-TOS (Vα-TOS)

Vα-TOS was generated as previously described [19]. Forty milligrams of α-TOS was dissolved in chloroform and a thin film was formed on the inside of a silanized 50 ml round-bottom flask by rotary evaporation under a nitrogen atmosphere (N2) and dried overnight in a desiccator. Approximately 1.9 ml of PBS (10 mM, pH 8.0) was added to the dry thin film and sonicated for 25 min in a water-bath sonicator (Branson 3510, Branson Ultrasonic Corp. Danbury, CT). Subsequently, 80 μl of 1 M NaOH was added to a final concentration of 40 mM and the suspension was sonicated for 20 min. Twenty microliter of 1 M HCl was added to a final concentration of 8 mM before a final sonication for 30 min. The resultant solution (20 mg/ml Vα-TOS) was used for in vitro and in vivo experiments. The vesicles of α-TOS generated ranged in size from 25 nm to 300 nm with 75% of the vesicles being smaller than 60 nm as determined by transmission electron microscopic analysis of negatively stained samples (data not shown).

Cell culture

The murine Lewis lung carcinoma cell line 3LLD122 (metastatic clone) was kindly provided by Dr. Lea Eisenbach (Weizmann Institute of Science, Rehovot, Israel). The cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) with 10% FBS. For DC culture, bone marrow (BM) cells were harvested from flushed marrow cavities of femurs and tibiae of C57BL/6 mice under aseptic conditions and cultured with 100 U/ml GM-CSF and 100 U/ml IL-4 at 106 cells/ml in complete media (RPMI + 10% heat inactivated FBS) as previously described [18]. On day 6, the non-adherent and loosely adherent cells were collected, washed with PBS before injecting 2×106 cells s.c. in mice. Dendritic cells were identified by FACS analysis on the basis of their expression of CD11c [18]. These cells were 50–60% positive for CD11c expression, 70–80% positive for MHC class II (I-Ab) expression. Of the CD11c+ cells, 2% were CD40 positive, 55% were CD80 positive and 38% were CD86 positive. To obtain mature DCs, day 6 DC were incubated with 200 U/ml TNF-α for 48 h [18]. Of the CD11c+ cells, 15%, 85% and 50% of the cells were positive for the expression of CD40, CD80 and CD86, respectively.

Vα-TOS treatment and assessment of tumor cell viability, clonogenic potential and apoptotic cell death

For the in vitro cell viability and apoptosis assays, 3LL tumor cells were plated at 2.5×105 cells/well in 6-well tissue culture dishes overnight. The cells were then treated with 0 μg/ml (PBS or 0.1% ethanol), 5 μg/ml, 10 μg/ml, 20 μg/ml, 40 μg/ml, 60 μg/ml or 80 μg/ml of Vα-TOS (in PBS) or α-TOS (in ethanol). After a 24 h exposure, non-adherent and adherent cells were collected and centrifuged at 200×g for 5 min. Cell number and viability were determined by trypan blue dye exclusion. For the clonogenicity assay, 102, 103, 104, and 105 viable cells from each treatment group (PBS, ethanol, 20 μg/ml, 40 μg/ml, 60 μg/ml of Vα-TOS or α-TOS) were plated in triplicate in 100-mm tissue culture dishes and incubated (7% CO2, 37°C) for 10 days in IMDM with 10% FBS. The resulting colonies were fixed in methanol and stained with Giemsa. Colonies containing >50 cells were counted and the surviving cell fraction was determined using the following formula: Surviving fraction = (# of colonies counted at a given concentration of Vα-TOS or α-TOS / # of cells plated at that concentration) / (# of control colonies counted (PBS or ethanol) / # of control cells plated) [21].

For the apoptosis assay, tumor cells were treated with either 40 μg/ml Vα-TOS or PBS. After 4, 12, or 18 h, non-adherent and adherent cells were collected and stained with Annexin V-FITC/PI following the manufacturer’s protocol (Roche Applied Sciences). Briefly, tumor cells were re-suspended in Annexin V binding buffer and stained with Annexin V-FITC and PI for 20 min in the dark. Binding buffer was added to the samples prior to flow cytometric analysis using the FACStarPLUS flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). The cells were gated on forward versus side scatter, and bivariate scattergrams of Annexin versus PI fluorescence were generated for analysis.

Co-culture of dendritic cells and tumor cells pre-treated with Vα-TOS

3LL tumor cells were plated at 2×105 cells/well in 6-well tissue culture plates in IMDM with 10% FBS. Twenty-four hours later, culture medium was removed and replaced with fresh medium containing 40 μg/ml Vα-TOS or PBS alone. After 24 h the supernatant fluid containing the non-adherent cells was collected and centrifuged at 22,600×g for 45 min. The pellet obtained was re-suspended in complete media and co-incubated with non-matured DC for 24 h in 12-well plates. 3LL tumor cell lysate generated by freeze-thaw (4 cycles) was added to a set of non-matured DC as a control. DCs were collected 24 h later, phenotyped and evaluated for IL-12p70 production. For the phenotypic analysis, DCs were collected and washed with PBS and stained for the expression of CD11c, I-Ab and the co-stimulatory molecules CD40, CD80 and CD86. To detect IL-12p70 production, 5×105 DCs were stimulated with 20 ng/ml TNF-α for 24 h in 48-well plates. The supernatant was collected after 24 h and analyzed for IL-12p70 production by ELISA according to the manufacturer’s protocol (Pierce Biotechnologies, Rockford, IL). In order to block the hsp receptor CD91, non-matured DC were incubated with or without 100 μg/ml of α2M, a natural ligand of CD91 [6], for 1 h in serum-free medium before addition of supernatant fluid from tumor cells treated with Vα-TOS. DCs were collected after 24 h and stained for the expression of CD11c, I-Ab, CD40, CD80 and CD86.

Expression of heat shock proteins by Vα-TOS-treated tumor cells

3LL cells were plated at 2×105 cells/well in 6-well tissue culture dishes. Twenty-four hours later, the medium was replaced with fresh culture medium containing 40 μg/ml of Vα-TOS or PBS alone and incubated for 12 h (7% CO2, 37°C). Non-adherent and adherent cells were collected, centrifuged at 200×g for 5 min and washed twice with PBSB. The cells were then re-suspended in PBSB and labeled with monoclonal antibodies specific for hsp60, 70 and 90, respectively for 45 min on ice. Controls included unlabeled cells and cells labeled with isotype IgG antibody. Cells were washed twice and stained with ALEXA FLOUR 488-conjugated goat anti-mouse secondary antibody for 45 min on ice. The cells were washed twice with PBSB before being finally re-suspended in PBSB for flow cytometric analysis.

Western Blot for hsp expression

3LL tumor cells were plated at 2×105 cells/well in 6-well tissue culture plates for 24 h in IMDM with 10% FBS. Twenty-four hours later, the culture medium was replaced with fresh medium containing 40 μg/ml Vα-TOS. After an additional 24 h, the non-adherent cells were collected and centrifuged (22,600×g, 45 min). The cell pellets were lysed using RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 μg/ml aproptinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin). The lysate was placed on a rocker at 4°C for 15 min and then forced 5 times through 25 gauge needles. The lysate was centrifuged at 14,000×g for 15 min at 4°C; the resultant supernatant was recovered and the protein content was determined using the BCA Protein Assay (Pierce Biotechnlogies, Rockford, IL). Proteins (30 μg) from the lysates were resolved by 10% SDS-PAGE and electro-transferred to polyvinylidene difluoride (PVDF) membrane. Nonspecific binding sites were blocked by incubating the membrane in TBST/MLK (Tris buffered saline containing 0.1% Tween-20 and 5% non-fat dry milk). The membrane was immunoblotted using mouse antibodies against either hsp60 (1:1000), hsp70 (1:500) or hsp90 (1:1000) (Stressgen Biotechnologies, Canada) and visualized with a goat anti-mouse HRP-conjugated secondary antibody (Santa Cruz Biotechnology, CA) using the Supersignal West Pico Chemiluminescent Substrate (Pierce Biotechnlogies, Rockford, IL).

Animal studies

Six-week-old female C57BL/6 mice were purchased from The Harlan Sprague Dawley Laboratory (Indianapolis, IN). Mice were housed at the University of Arizona Animal Facilities in accordance with the Principles of Animal Care (NIH publication No. 85-23, revised 1985). For establishment of primary tumors, each mouse was injected s.c. with 106 3LLD122 tumor cells in 50 μl PBS on the right hind flank. After tumors were established (25–30 mm3) on day 9 or 10, the mice were randomized based on tumor volume and subjected to different treatment regimens. Mice were given 9 i.p. injections of Vα-TOS (4 mg/injection in 200 μl of PBS at 200 mg/kg body weight) on alternate days starting on day 9 or 10 after tumor cell injection. The control group consisted of mice injected with 200 μl of PBS. For the combination treatment, 1×106 DC were injected s.c. on days 12, 16, and 20 in 50 μl of PBS. Tumor growth was monitored by measuring the tumor length and width with calipers and calculating the tumor volume according to the formula V= (L×W 2)/2 [34].

In vivo quantification of α-TOS and α-TOH

Serum and tissue levels of α-TOS and α-TOH were determined by the Analytical Core Facility of the Arizona Cancer Center using a modification of a previously described method [12]. Serum was obtained by retro-orbital bleeding or terminal heart bleed and 5 nmol of α-tocopheryl acetate (α-TOA) was added as internal standard. Subsequently, the samples were extracted twice with 0.5 ml hexane and evaporated to dryness under nitrogen. For α-TOS levels in tumor tissue, 10 mg of tumor tissue was minced and incubated for 1 h at 37°C with 0.3 ml of enzyme mix (5 mg/ml collagenase and 2 mg/ml pronase E). Next, 0.3 ml of 1% sodium dodecyl sulfate in ethanol with 0.1% butylated hydroxy-toluene were added to the samples. The samples were extracted twice with 0.6 ml of hexane and evaporated to dryness under nitrogen. The residues obtained after drying the serum or tissue hexane extracts were dissolved in 100 μl methanol and analyzed by high-performance liquid chromatography.

Statistical analysis

Statistical significance of differences among data sets of treatment groups were assessed by one-way analysis of variances (ANOVA) including Tukey-Kramer post tests for multiple comparisons. Log-rank tests were performed on the Kaplan-Meier survival curves of Vα-TOS+/- DC-treated and control (sham-treated) animals. Decline in serum α-TOS levels were evaluated by linear regression analysis including runs-test. All analyses were performed using the Prism software (GraphPad, San Diego, CA). Probability values (P) of ≤0.05 were considered indicative of significant differences between data sets.

Results

Vα-TOS is toxic to tumor cells in vitro

We evaluated the in vitro tumoricidal activities of Vα-TOS and α-TOS on 3LL tumor cells. For this purpose, tumor cells were exposed to different concentrations of Vα-TOS and α-TOS for 24 h and viable cell number determined by trypan blue dye exclusion. In addition, the clonogenic potential of surviving cells was determined. The data show that both Vα-TOS and α-TOS kill 3LL tumor cells in a dose-dependent manner (Fig. 1a). The differences between α-TOS and Vα-TOS induced cell death were statistically significant at 10 μg/ml (P<0.0077), 20 μg/ml (P<0.0221), 40 μg/ml (P<0.0005), and 80 μg/ml (P<0.0012). The IC50 values of Vα-TOS and α-TOS were 18 μg/ml and 30 μg/ml, respectively. In addition, tumor cells that survived the 24-h Vα-TOS or α-TOS treatment were significantly impaired in their ability to proliferate and form colonies (Fig. 1b) with Vα-TOS being more toxic than α-TOS at doses of 20 μg/ml (P<0.0022), and 40 μg/ml (P<0.0004), respectively. Exposure to Vα-TOS also induced 3LL cells to undergo apoptosis as a function of time (Fig. 1c) as described earlier for α-TOS [29]. Vα-TOS-induced phosphatidyl serine translocation to the cell surface signifying early apoptosis (Annexin V positive) was observed at 4 h and progressively increased with time leading to secondary loss of membrane integrity (Annexin V and PI positive) by 18 h.

Fig. 1.

Fig. 1

Open in a new tab

Effect of Vα-TOS on tumor cell viability in vitro. 3LL cells were allowed to adhere overnight in 6-well tissue culture plates at 2.5×105 cells per well in triplicate. The cells were then treated with 0 μg/ml (PBS or 0.1% ethanol), 5 μg/ml, 10 μg/ml, 20 μg/ml, 40 μg/ml, 60 μg/ml or 80 μg/ml of Vα-TOS (in PBS) or α-TOS (in ethanol). After a 24-h exposure, non-adherent and adherent cells were collected and cell number and viability were determined by trypan blue dye exclusion. The data (A) are representative of two independent experiments and the values denote means ± SD of triplicate samples. In order to determine the clonogenic potential of the cells (B) 102, 103, 104, and 105 viable cells from each treatment group were plated in triplicate in 100-mm tissue culture dishes and incubated (7% CO2, 37°C) for 10 days in culture medium. The resulting colonies were fixed and Giemsa stained. Colonies containing >50 cells were counted and the surviving cell fraction was determined as described in Materials and Methods. The values represent means ± SD of triplicate samples. For the apoptosis assay (C) cells were treated with either 40 μg/ml Vα-TOS or PBS. At each time point, non-adherent and adherent cells were collected and stained using Annexin V and PI. Numbers represent the percentages of early apoptotic cells (lower right quadrant) and secondary necrotic cells (upper right quadrant), respectively

Alpha-TOS remains in the active form after intraperitoneal injection of Vα-TOS

One of our concerns was whether Vα-TOS remains in its active form or is de-esterified to inactive alpha-tocopherol (α-TOH) in vivo. To address this concern, Vα-TOS was administered i.p. and plasma and tumor tissue levels of α-TOS and α-TOH were determined after 24, 48, 72 h (plasma) or 48 h (tumor tissue) by HPLC. The data show that Vα-TOS can be readily measured in plasma and tumor tissue and that Vα-TOS is minimally de-esterified to α-TOH (Fig. 2a, b). The data also demonstrate that the drug remains in the active succinate form in the serum, with a half-life of approximately 35 h and declines in a linear fashion (Fig. 2a). Regression analysis including runs-test revealed a significantly non-zero slope.

Fig. 2.

Fig. 2

Open in a new tab

Plasma and tumor tissue levels of α-TOS and α-TOH after i.p. administration of Vα-TOS. Mice were injected with 200 μl (4 mg) of Vα-TOS or 200 μl of PBS. A After 24, 48 and 72 h, blood was collected by eye bleed and plasma levels (pooled from two mice) of α-TOS and α-TOH were determined by HPLC. B Mice were injected with tumor cells. After tumor development, the mice were injected with 200 μl (4 mg) of Vα-TOS or 200 μl of PBS. Forty-eight hours after Vα-TOS injection, the mice were sacrificed and the tumors removed. Hexane extracts of the tumor tissues were prepared (pooled from two mice) to determine α-TOS and α-TOH levels by HPLC

Vα-TOS is as effective as α-TOS at inhibiting 3LL tumor growth in vivo

Having shown that Vα-TOS kills tumor cells in vitro, we evaluated its ability to control the growth of established 3LL tumors in vivo. For this purpose, animals bearing established tumors were given nine sequential injections of Vα-TOS or α-TOS and tumor growth was assessed. The data demonstrate that Vα-TOS and α-TOS significantly (P<0.05) inhibit the growth of pre-established 3LL tumors (Fig. 3). Tumors grew unchecked in the vitamin E (α-TOH)-injected and control mice (ethanol, PBS) and ranged in size from 1049.8±246.2 mm3 to 1269.3±201.7 mm3 on day 28 post-tumor injection. In contrast, tumor volumes in mice injected with Vα-TOS or α-TOS were 379.7±44.1 mm3 and 438.4±161.6 mm3, respectively. The in vivo tumor suppressive activity of Vα-TOS has also been demonstrated by us in a poorly immunogenic murine breast cancer model (Ramanathapuram et al., submitted).

Fig. 3.

Fig. 3

Open in a new tab

Effect of Vα-TOS on growth of pre-established 3LL tumors. Mice were injected s.c. with 106 3LL tumor cells. On development of palpable tumors (day 10) mice were injected (i.p.) with either 4 mg of Vα-TOS or α-TOS starting on day 10 at 2-day intervals for a total of nine injections. The data show mean tumor volumes ± SD of seven individual mice per group

Non-matured DCs are as effective as matured DCs in combination with Vα-TOS at inhibiting tumor growth

In an earlier study [29], we showed that α-TOS potentiates the effect of adoptively transferred non-matured DC in treating pre-established 3LL tumors. In order to ascertain if mature DCs are as effective as non-matured DCs in combination with Vα-TOS, we treated mice bearing pre-established 3LL tumors with a combination of Vα-TOS (i.p.) + either non-matured DCs (nmDC) or DCs matured with TNF-α (mDC) (s.c.). The data (Fig. 4a) demonstrate that when used in combination with Vα-TOS, nmDCs are as effective as mDCs at inhibiting the growth of 3LL tumors (P>0.05). The mean tumor volumes in mice receiving Vα-TOS plus either nmDC or mDC were 322.7±123.9 mm3 and 367.8±61.7 mm3 respectively on day 29 post-tumor cell injection and were significantly lower (P<0.001) than the mean tumor volume of 769.8±187.9 mm3 in mice receiving Vα-TOS alone. The data also show that injection of Vα-TOS plus DC prolongs the survival of mice (P<0.0001) as compared to mice injected with Vα-TOS alone (Fig. 4b). However, there was no statistically significant prolongation of survival of mice treated with Vα-TOS alone compared to mice treated with PBS+nmDC or PBS+mDC (P>0.05). All control animals died because of large tumor burden (∼2,000 mm3) by day 36 and mice injected with Vα-TOS died naturally or were sacrificed when tumor volumes reached ∼2000 mm3 by day 40. In contrast, mice in the Vα-TOS + nmDC/mDC groups were alive until day 48 when they were terminated as tumor volumes reached ∼2,000 mm3.

Fig. 4.

Fig. 4

Open in a new tab

Effect of Vα-TOS plus DC immunotherapy on pre-established 3LL tumors. Mice were injected s.c. with 106 3LL tumor cells. On development of palpable tumors (day 9), mice were injected i.p. with 4 mg of Vα-TOS on alternate days for a total of nine injections. The mice were also injected s.c. with either 106 non-matured DC (nmDC), or non-antigen pulsed, TNF-α matured DC (mDC) on days 12, 16 and 20. The data represent A mean tumor volumes ± SD and B % survival of six individual mice per group. All control animals died because of large tumor burden or were sacrificed when tumor volume was ∼2,000 mm3 by day 36. Mice injected with Vα-TOS died naturally or were sacrificed when tumors reached a size of ∼2000 mm3 (day 40). Mice in the Vα-TOS + nmDC/mDC groups were alive until day 48 when they were terminated due to large tumor burden (∼2,000 mm3)

Vα-TOS-treated tumor cells induce maturation of DCs in vitro

Having shown that nmDC plus Vα-TOS inhibit tumor growth as effectively as mDC plus Vα-TOS, we hypothesized that Vα-TOS-treated tumor cells cause DC maturation. To examine this possibility, we incubated nmDC with supernatant derived from tumor cells exposed to Vα-TOS for 24 h and assessed expression of maturation markers (CD40, CD80, CD86). The data (Fig. 5) show that co-incubation of supernatant of Vα-TOS-treated tumor cells with nmDC causes up-regulation of the co-stimulatory molecules, CD40, CD80 and CD86 on DC as evidenced by mean fluorescence intensity changes (ΔMFI) of 15, 100 and 34, respectively compared to untreated nmDC. These changes induced by Vα-TOS-treated tumor cells were comparable to those caused by TNF-α, a cytokine commonly used to mature DC (data not shown). In contrast, direct incubation of nmDC with Vα-TOS or nmDC with freeze-thawed tumor lysate (data not shown) for the same length of time did not cause an increase in the expression of these markers above background (nmDC alone or nmDC incubated with supernatant from PBS-treated tumor cells). The DC collected after incubation with supernatant from Vα-TOS-treated tumor cells were also evaluated for the production of IL-12p70. The data (Fig. 6) show that nmDC incubated with Vα-TOS-treated tumor cells produced 202.4±15.5 pg/ml of IL-12p70, which was significantly higher (P<0.001) than that secreted by nmDC alone (63.8±13.9 pg/ml), nmDC incubated with supernatant from PBS-treated tumor cells (60±6.2 pg/ml) or nmDC incubated with tumor cell lysate (67.3±6.5 pg/ml).

Fig. 5.

Fig. 5

Open in a new tab

Effect of Vα-TOS treated tumor cells on expression of co-stimulatory markers on DCs. 3LL cells were treated with 40 μg/ml Vα-TOS or PBS for 24 h. The supernatant was collected and centrifuged at 22,600×g for 45 min to collect non-adherent cells and membrane debris. The pellet obtained was re-suspended in media and incubated with non-matured bone marrow-derived DCs for 24 h. DCs were collected and stained with PE-conjugated CD11c antibody and FITC-conjugated antibodies against CD40, CD80 and CD86 and analyzed by flow cytometry. DC represents untreated DC, DC+PBSs represents DC incubated with supernatant from PBS-treated 3LL cells; DC+Vα-TOSs represents DC incubated with supernatant from Vα-TOS-treated 3LL cells; DC+Vα-TOS represents DC treated with 40 μg/ml Vα-TOS. The data are representative of three independent experiments

Fig. 6.

Fig. 6

Open in a new tab

Effect of Vα-TOS treated tumor cells on IL-12p70 production by non-matured DCs. 3LL cells were treated with 40 μg/ml Vα-TOS or PBS for 24 h. The supernatant was collected and centrifuged at 22,600×g for 45 min. The pellet obtained was re-suspended in media and added to non-matured DCs for 24 h. DCs were then re-stimulated with TNF-α for 24 h in 48-well tissue culture plates after which the supernatant was collected and evaluated for IL-12p70 production by ELISA. DC represents untreated DC, DC+PBSs represents DC incubated with supernatant from PBS-treated 3LL cells; DC+Vα-TOSs represents DC incubated with supernatant from Vα-TOS-treated 3LL cells; DC+Vα-TOS represents DC treated with 40 μg/ml Vα-TOS, DC+lysate represents DC incubated with freeze-thaw lysate of 3LL tumor cells. The data are representative of three independent experiments and denote mean ± SD of triplicate samples

Vα-TOS induces expression of heat shock proteins on tumor cells

Heat shock proteins (hsps) are well-documented to be up-regulated by stress [25, 31] and have been suggested to provide “danger signals” that lead to the activation/maturation of dendritic cells [7, 9, 10, 13, 14]. Since Vα-TOS-treated tumor cells caused DC maturation, we hypothesized that hsps might be involved in the process. To address the possibility that Vα-TOS treatment up-regulates hsp expression in tumor cells, 3LL cells were exposed to 40 μg/ml Vα-TOS for 12 h, stained with monoclonal antibodies specific for hsp60, 70 and 90 and analyzed by flow cytometry. The data (Fig. 7a) show that Vα-TOS induces membrane expression of heat shock proteins 60, 70 and 90 on tumor cells as compared to tumor cells treated with vehicle (PBS). The differential induction of hsps was also confirmed by Western blot analysis of supernatant fluid derived from Vα-TOS-treated cells (Fig. 7b). A similar induction of these hsps has been demonstrated in 4T1 murine mammary tumor cells treated with Vα-TOS (Ramanathapuram et al., submitted).

Fig. 7.

Fig. 7

Open in a new tab

Heat shock protein expression in tumor cells after treatment with Vα-TOS. 3LL cells were treated with either 40 μg/ml Vα-TOS or PBS (vehicle). After 12 h, non-adherent and adherent cells were collected, washed twice with PBS and stained with antibodies against hsp60, 70 or 90. Goat anti-mouse IgG-ALEXA-FLOUR 488 was used as the secondary antibody. A Flow cytometric analysis was performed on intact cells based on light scatter gates. The data are representative of two independent experiments. Shaded box represents PBS-treated cells and solid line represents Vα-TOS-treated cells. B 3LL cells were treated with either 40 μg/ml Vα-TOS or PBS for 24 h. Supernatant was then collected and centrifuged at 22,600×g for 45 min. The pellet obtained was lysed, protein concentration measured and separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked using 5% non-fat dry milk and stained with hsp60, 70 and 90-specific antibodies, respectively. The membranes were washed, and stained with a goat anti-mouse HRP-conjugated secondary antibody before visualization by chemiluminescence. Vα-TOSs represents lysate derived from Vα-TOS-treated 3LL tumor supernatant, PBSs represents lysate derived from PBS-treated 3LL tumor supernatant

Pre-treatment of DC with α2-macroglobulin partially inhibits Vα-TOS induced expression of co-stimulatory molecules

Since Vα-TOS treatment leads to hsp expression in tumor cells, we hypothesized that these hsps are involved in the maturation of DCs observed in vitro. In order to further address this possibility, nmDC were pre-treated with α2-macroglobulin (α2M) to block the cognate hsp receptor CD91 prior to incubation with supernatant derived from tumor cells exposed to Vα-TOS for 24 h. The data (Fig. 8) show that pre-treatment with α2M, followed by addition of supernatant derived from Vα-TOS treated tumor cells partially inhibited the expression of the maturation markers CD40, CD80 and CD86 on DCs. Incubation of DC with α2M alone, did not cause any change in the expression of maturation markers.

Fig. 8.

Fig. 8

Open in a new tab

Effect of pre-treatment of non-matured DCs with α2-macroglobulin on maturation induced by Vα-TOS treated tumor cells. Non-matured DCs were incubated in serum-free media with or without 100 μg/ml α2M for an hour. 3LL cells were treated with 40 μg/ml Vα-TOS or PBS for 24 h. The supernatant was collected and centrifuged at 22,600×g for 45 min. The pellet obtained was re-suspended in media and added to the pre-treated DC for 24 h. DCs were collected and stained with PE-conjugated CD11c antibody and FITC-conjugated antibodies against CD40, CD80 and CD86 and analyzed by flow cytometry. DC represents untreated DC; DC+α2M represents DC pre-treated with α2M, DC+Vα-TOSs represents DC incubated with supernatant from Vα-TOS-treated 3LL cells, DC+α2M+Vα-TOSs represents DC pre -treated with α2M and incubated with supernatant from Vα-TOS-treated 3LL cells. The data are representative of two independent experiments

Discussion

In a previous study, we demonstrated that α-TOS synergizes with non-antigen pulsed, non-matured DCs in vivo to mediate growth suppression of a murine Lewis lung carcinoma (3LL tumors) [29]. In this study, we have used vesiculated α-TOS (Vα-TOS) in combination with non-antigen pulsed, non-matured or matured dendritic cells to treat established 3LL tumors. Unlike the liposomal α-TOS generated by sonication of α-TOS and mixing with lipids [2], vesiculated α-TOS is formed by spontaneous self assembly of α-TOS and is more soluble than α-TOS in aqueous solutions [17, 19]. We observed that aqueous Vα-TOS is as effective as α-TOS dissolved in ethanol at inhibiting tumor cell growth in vitro and in vivo. We have also demonstrated that non-matured DCs were just as effective as non-antigen pulsed TNFα-matured DCs at controlling tumor growth when either one is combined with Vα-TOS in a treatment regimen. The in vivo anti-tumor activity of Vα-TOS could have been somewhat mitigated by its susceptibility to host-derived esterases that convert α-TOS to the apoptosis-inert, parent molecule, alpha-tocopherol (α-TOH). This likelihood is supported by recent reports by Kline’s group [1, 20] demonstrating the superior anti-tumor activity of a non-hydrolyzable ether analog of vitamin E, α-TEA over α-TOS. When administered as an aerosol, a liposomal formulation of α-TEA was more effective than α-TOS at suppressing primary tumor growth and reducing lung metastases in a murine breast cancer model [1, 20]. Ongoing studies in our laboratory are evaluating the potencies of Vα-TEA and Vα-TOS in lung and mammary tumor models.

Unlike the majority of DC-based vaccine strategies, our approach eliminates the requirement for additional ex-vivo manipulations such as maturation and/or loading of DC with tumor antigens in order to generate DC capable of mediating anti-tumor activity in vivo. This finding suggests that Vα-TOS treatment of tumor cells causes DC maturation, rendering non-matured DC as effective as TNF-α matured DC in mediating tumor suppression. This possibility is supported by our finding that co-incubation of DC with supernatant fluid derived from Vα-TOS-treated cells leads to up-regulation of the co-stimulatory molecules, CD40, CD80 and CD86 and increased secretion of IL-12p70. Previous studies have shown that exposure of DC to stressed apoptotic tumor cells and lysates or supernatants of necrotic transformed cell lines leads to maturation of human and murine dendritic cells [7, 13, 30, 32, 37]. Since Vα-TOS also induces apoptosis of tumor cells leading to secondary necrosis, its ability to mediate DC maturation is consistent with published reports.

In further trying to elucidate the mechanism by which Vα-TOS may cause DC maturation, we demonstrated that Vα-TOS induces the expression of heat shock proteins 60, 70 and 90 in the tumor cells. Heat shock proteins are one of the most abundant soluble intracellular molecules that function as molecular chaperones. They have essential roles in protecting cells from potentially lethal effects of stress and proteotoxicity [25]. The presence of hsps in the extracellular environment acts as a “danger signal” that alerts antigen presenting cells including DC of potential damage or infection leading to their activation [13, 25, 30, 31]. This conversion makes DCs effective antigen presenters which migrate to secondary lymphoid organs where they initiate anti-tumor T cell responses [4]. Heat shock protein 60, 70 and 90 have been shown to induce the maturation of DC [7, 8, 9, 14, 32] and up-regulate the expression of pro-inflammatory cytokines [3, 10, 16]. In this study, we also observed that co-incubation of DC with α2-macroglobulin which competes with hsp60, 70 and 90 for binding to the cognate receptor CD91 caused a partial reduction in the expression of co-stimulatory molecules induced by supernatant fluid derived from Vα-TOS-treated cells. These results suggest potential involvement of hsps in Vα-TOS-mediated DC activation. The absence of complete inhibition of co-stimulatory molecule expression suggests involvement of additional hsp receptors and/or hsps including gp96 and calreticulin. Experiments are ongoing to investigate this possibility.

Taken together, these results lead us to postulate that Vα-TOS employs at least a two-pronged approach to potentiate DC-mediated immunotherapy of cancer; firstly, by direct killing of tumor cells whose antigens can be cross-presented by DC and secondly by maturation of DC via hsp-mediated “danger signals”. Additional studies are needed to confirm this hypothesis. Notwithstanding, the combination of Vα-TOS and DC appears to be a potentially useful therapeutic strategy to control established tumors that can be translated to the clinic.

Acknowledgements

We would like to thank Barbara Carolus and Debbie Sakiestewa for flow cytometric analysis. We would also like to thank Dr. Steve Stratton and Min-Jian Xu of the Analytical Core Facility (supported by the grant 5 P30 CA23074) at the Arizona Cancer Center for HPLC analysis.

Abbreviations

DC

Dendritic cell

α-TOS

Alpha-tocopheryl succinate

α2M

Alpha-2 macroglobulin

IFN-γ

Interferon-gamma

i.p.

Intraperitoneal

s.c.

Subcutaneous

FBS

Fetal bovine serum

PBS

Phosphate buffered saline

PBSB

Phosphate buffered saline with bovine serum albumin

hsp

Heat shock protein

GM-CSF

Granulocyte/macrophage colony-stimulating factor

IL-4

Interleukin-4

IL-12p70

Interleukin-12

TNF-α

Tumor necrosis factor-alpha

FACS

Fluorescence-activated cell sorter

PI

Propidium iodide

FITC

Fluorescein isothiocyanate

HRP

Horse radish peroxidase

Footnotes

Supported by Grants 1 RO1 CA94111-02 from the NIH and DAMD 17010126 from the DOD.

References

  • 1.Anderson K, Lawson KA, Simmons-Menchaca M, Sun L, Sanders BG, Kline K. Alpha-TEA plus cisplatin reduces human cisplatin-resistant ovarian cancer cell tumor burden and metastasis. Exp Biol Med (Maywood) 2004;229:1169–1176. doi: 10.1177/153537020422901112. [DOI] [PubMed] [Google Scholar]
  • 2.Anderson K, Simmons-Menchaca M, Lawson KA, Atkinson J, Sanders BG, Kline K. Differential response of human ovarian cancer cells to induction of apoptosis by vitamin E Succinate and vitamin E analogue, alpha-TEA. Cancer Res. 2004;64:4263–4269. doi: 10.1158/0008-5472.CAN-03-2327. [DOI] [PubMed] [Google Scholar]
  • 3.Asea A, Kraeft SK, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, Koo GC, Calderwood SK. HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nat Med. 2000;6:435–442. doi: 10.1038/74697. [DOI] [PubMed] [Google Scholar]
  • 4.Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245–252. doi: 10.1038/32588. [DOI] [PubMed] [Google Scholar]
  • 5.Barnett KT, Fokum FD, Malafa MP. Vitamin E succinate inhibits colon cancer liver metastases. J Surg Res. 2002;106:292–298. doi: 10.1006/jsre.2002.6466. [DOI] [PubMed] [Google Scholar]
  • 6.Basu S, Binder RJ, Ramalingam T, Srivastava PK. CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity. 2001;14:303–313. doi: 10.1016/S1074-7613(01)00111-X. [DOI] [PubMed] [Google Scholar]
  • 7.Basu S, Binder RJ, Suto R, Anderson KM, Srivastava PK. Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-kappa B pathway. Int Immunol. 2000;12:1539–1546. doi: 10.1093/intimm/12.11.1539. [DOI] [PubMed] [Google Scholar]
  • 8.Basu S, Srivastava PK. Fever-like temperature induces maturation of dendritic cells through induction of hsp90. Int Immunol. 2003;15:1053–1061. doi: 10.1093/intimm/dxg104. [DOI] [PubMed] [Google Scholar]
  • 9.Bethke K, Staib F, Distler M, Schmitt U, Jonuleit H, Enk AH, Galle PR, Heike M. Different efficiency of heat shock proteins (HSP) to activate human monocytes and dendritic cells: superiority of HSP60. J Immunol. 2002;169:6141–6148. doi: 10.4049/jimmunol.169.11.6141. [DOI] [PubMed] [Google Scholar]
  • 10.Chen W, Syldath U, Bellmann K, Burkart V, Kolb H. Human 60-kDa heat-shock protein: a danger signal to the innate immune system. J Immunol. 1999;162:3212–3219. [PubMed] [Google Scholar]
  • 11.Dalen H, Neuzil J. Alpha-tocopheryl succinate sensitises a T lymphoma cell line to TRAIL-induced apoptosis by suppressing NF-kappaB activation. Br J Cancer. 2003;88:153–158. doi: 10.1038/sj.bjc.6600683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Fariss MW, Merson MH, O’Hara TM. Alpha-tocopheryl succinate protects hepatocytes from chemical-induced toxicity under physiological calcium conditions. Toxicol Lett. 1989;47:61–75. doi: 10.1016/0378-4274(89)90086-6. [DOI] [PubMed] [Google Scholar]
  • 13.Feng H, Zeng Y, Graner MW, Katsanis E. Stressed apoptotic tumor cells stimulate dendritic cells and induce specific cytotoxic T cells. Blood. 2002;100:4108–4115. doi: 10.1182/blood-2002-05-1389. [DOI] [PubMed] [Google Scholar]
  • 14.Flohe SB, Bruggemann J, Lendemans S, Nikulina M, Meierhoff G, Flohe S, Kolb H. Human heat shock protein 60 induces maturation of dendritic cells versus a Th1-promoting phenotype. J Immunol. 2003;170:2340–2248. doi: 10.4049/jimmunol.170.5.2340. [DOI] [PubMed] [Google Scholar]
  • 15.Israel K, Yu W, Sanders BG, Kline K. Vitamin E succinate induces apoptosis in human prostate cancer cells: role for Fas in vitamin E succinate-triggered apoptosis. Nutr Cancer. 2000;36:90–100. doi: 10.1207/S15327914NC3601_13. [DOI] [PubMed] [Google Scholar]
  • 16.Jin Y, Fuller L, Ciancio G, Burke GW, III, Tzakis AG, Ricordi C, Miller J, Esquenzai V. Antigen presentation and immune regulatory capacity of immature and mature-enriched antigen presenting (dendritic) cells derived from human bone marrow. Hum Immunol. 2004;65:93–103. doi: 10.1016/j.humimm.2003.11.002. [DOI] [PubMed] [Google Scholar]
  • 17.Jizomoto H, Kanaoka E, Hirano K. pH-sensitive liposomes composed of tocopherol hemisuccinate and of phosphatidylethanolamine including tocopherol hemisuccinate. Biochim Biophys Acta. 1994;1213:343–348. [PubMed] [Google Scholar]
  • 18.Kobie JJ, Wu RS, Kurt RA, Lou S, Adelman MK, Whitesell LJ, Ramanathapuram LV, Arteaga CL, Akporiaye ET. Transforming growth factor beta inhibits the antigen-presenting functions and antitumor activity of dendritic cell vaccines. Cancer Res. 2003;63:1860–1864. [PubMed] [Google Scholar]
  • 19.Kogure K, Manabe S, Hama S, Tokumura A, Fukuzawa K. Potentiation of anti-cancer effect by intravenous administration of vesiculated alpha-tocopheryl hemisuccinate on mouse melanoma in vivo. Cancer Lett. 2003;192:19–24. doi: 10.1016/S0304-3835(02)00683-3. [DOI] [PubMed] [Google Scholar]
  • 20.Lawson KA, Anderson K, Menchaca M, Atkinson J, Sun L, Knight V, Gilbert BE, Conti C, Sanders BG, Kline K. Novel vitamin E analogue decreases syngeneic mouse mammary tumor burden and reduces lung metastasis. Mol Cancer Ther. 2003;2:437–444. [PubMed] [Google Scholar]
  • 21.Lawson KA, Anderson K, Simmons-Menchaca M, Atkinson J, Sun L, Sanders BG, Kline K. Comparison of vitamin E derivatives alpha-TEA and VES in reduction of mouse mammary tumor burden and metastasis. Exp Biol Med (Maywood) 2004;229:954–963. doi: 10.1177/153537020422900913. [DOI] [PubMed] [Google Scholar]
  • 22.Liu B, Wu K, Zhao D. Study on vitamin E succinate inducing apoptosis of human gastric carcinoma cell. Wei Sheng Yan Jiu. 2000;29:234–236. [PubMed] [Google Scholar]
  • 23.Malafa MP, Fokum FD, Mowlavi A, Abusief M, King M. Vitamin E inhibits melanoma growth in mice. Surgery. 2002;131:85–91. doi: 10.1067/msy.2002.119191. [DOI] [PubMed] [Google Scholar]
  • 24.Malafa MP, Neitzel LT. Vitamin E succinate promotes breast cancer tumor dormancy. J Surg Res. 2000;93:163–170. doi: 10.1006/jsre.2000.5948. [DOI] [PubMed] [Google Scholar]
  • 25.Mosser DD, Morimoto RI. Molecular chaperones and the stress of oncogenesis. Oncogene. 2004;23:2907–2918. doi: 10.1038/sj.onc.1207529. [DOI] [PubMed] [Google Scholar]
  • 26.Neuzil J, Weber T, Gellert N, Weber C. Selective cancer cell killing by alpha-tocopheryl succinate. Br J Cancer. 2001;84:87–89. doi: 10.1054/bjoc.2000.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Neuzil J, Weber T, Terman A, Weber C, Brunk UT. Vitamin E analogues as inducers of apoptosis: implications for their potential antineoplastic role. Redox Rep. 2001;6:143–151. doi: 10.1179/135100001101536247. [DOI] [PubMed] [Google Scholar]
  • 28.Prasad KN, Kumar B, Yan XD, Hanson AJ, Cole WC. Alpha-tocopheryl succinate, the most effective form of vitamin E for adjuvant cancer treatment: a review. J Am Coll Nutr. 2003;22:108–117. doi: 10.1080/07315724.2003.10719283. [DOI] [PubMed] [Google Scholar]
  • 29.Ramanathapuram LV, Kobie JJ, Bearss D, Payne CM, Trevor KT, Akporiaye ET. alpha-Tocopheryl succinate sensitizes established tumors to vaccination with nonmatured dendritic cells. Cancer Immunol Immunother. 2004;53:580–588. doi: 10.1007/s00262-004-0499-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Somersan S, Larsson M, Fonteneau JF, Basu S, Srivastava P, Bhardwaj N. Primary tumor tissue lysates are enriched in heat shock proteins and induce the maturation of human dendritic cells. J Immunol. 2001;167:4844–4852. doi: 10.4049/jimmunol.167.9.4844. [DOI] [PubMed] [Google Scholar]
  • 31.Srivastava P. Interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses. Annu Rev Immunol. 2002;20:395–425. doi: 10.1146/annurev.immunol.20.100301.064801. [DOI] [PubMed] [Google Scholar]
  • 32.Todryk S, Melcher AA, Hardwick N, Linardakis E, Bateman A, Colombo MP, Stoppacciaro A, Vile RG. Heat shock protein 70 induced during tumor cell killing induces Th1 cytokines and targets immature dendritic cell precursors to enhance antigen uptake. J Immunol. 1999;163:1398–1408. [PubMed] [Google Scholar]
  • 33.Weber T, Lu M, Andera L, Lahm H, Gellert N, Fariss MW, Korinek V, Sattler W, Ucker DS, Terman A, Schroder A, Erl W, Brunk UT, Coffey RJ, Weber C, Neuzil J. Vitamin E succinate is a potent novel antineoplastic agent with high selectivity and cooperativity with tumor necrosis factor-related apoptosis-inducing ligand (Apo2 ligand) in vivo. Clin Cancer Res. 2002;8:863–869. [PubMed] [Google Scholar]
  • 34.Wu RS, Kobie JJ, Besselsen DG, Fong TC, Mack VD, McEarchern JA, Akporiaye ET. Comparative analysis of IFN-gamma B7.1 and antisense TGF-beta gene transfer on the tumorigenicity of a poorly immunogenic metastatic mammary carcinoma. Cancer Immunol Immunother. 2001;50:229–240. doi: 10.1007/s002620100197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Yu W, Sanders BG, Kline K. RRR-alpha-tocopheryl succinate inhibits EL4 thymic lymphoma cell growth by inducing apoptosis and DNA synthesis arrest. Nutr Cancer. 1997;27:92–101. doi: 10.1080/01635589709514508. [DOI] [PubMed] [Google Scholar]
  • 36.Yu W, Simmons-Menchaca M, Gapor A, Sanders BG, Kline K. Induction of apoptosis in human breast cancer cells by tocopherols and tocotrienols. Nutr Cancer. 1999;33:26–32. doi: 10.1080/01635589909514744. [DOI] [PubMed] [Google Scholar]
  • 37.Zheng H, Dai J, Stoilova D, Li Z. Cell surface targeting of heat shock protein gp96 induces dendritic cell maturation and antitumor immunity. J Immunol. 2001;167:6731–6735. doi: 10.4049/jimmunol.167.12.6731. [DOI] [PubMed] [Google Scholar]

Articles from Cancer Immunology, Immunotherapy : CII are provided here courtesy of Springer

RESOURCES