Exosomal delivery of berry anthocyanidins for the management of ovarian cancer

Farrukh Aqil *ab, Jeyaprakash Jeyabalan a, Ashish K. Agrawal a, Al-Hassan Kyakulaga c, Radha Munagala ab, Lynn Parker d and Ramesh C. Gupta *ac
aJames Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, USA. E-mail: farrukh.aqil@louisville.edu; rcgupta@louisville.edu; Fax: +502 852 3842; Tel: +502 852 3682
bDepartment of Medicine, University of Louisville, Louisville, KY 40202, USA
cDepartment of Pharmacology & Toxicology, University of Louisville, Louisville, KY 40202, USA
dNorton Cancer Institute, 315 E. Broadway, 4th Floor, Louisville, KY 40202, USA

Received 14th June 2017 , Accepted 6th September 2017

First published on 9th October 2017


Despite optimal diagnosis and early therapeutic interventions, the prognosis for ovarian cancer patients remains dismal because the efficacy of chemotherapy is limited by the development of resistance and off-site toxicity. Berry bioactives indicate preventive and therapeutic activities against various cancer types. Here, we examined the antiproliferative activity of berry anthocyanidins (Anthos) against drug-sensitive (A2780) and drug-resistant (A2780/CP70, OVCA432 and OVCA433) ovarian cancer cells. These drug-resistant ovarian cancer cell lines overexpress p-glycoproteins (PgP) and show >100-fold resistance to the chemotherapeutic drug cisplatin compared to A2780. We observed a dose-dependent growth inhibition of ovarian cancer cells with the Anthos. Furthermore, the treatment of drug-resistant ovarian cancer (OVCA433) cells with cisplatin in combination with the Anthos (75 μM) resulted in significantly higher cell killing. The cisplatin dose required to achieve this effect was 10 to 15-fold lower than the IC50 of cisplatin alone. However, many plant bioactives including Anthos face the challenge of poor oral bioavailability and stability. Recently, we have developed strategies to overcome these limitations by delivering Anthos via milk-derived exosomes. The exosomal Anthos (ExoAnthos) significantly enhanced the antiproliferative activity against the growth of ovarian cancer cells and inhibited tumor growth more efficiently compared to Anthos alone and a vehicle control. Often patients with cisplatin-resistant tumors retain sensitivity to paclitaxel (PAC). We prepared exosomal formulations of PAC (ExoPAC) for oral delivery as the systemic administration of PAC has severe side effects. ExoPAC delivered orally showed the same therapeutic efficacy as the free PAC delivered intraperitoneally. Finally, we report that the combination of the Anthos and PAC decreased the PgP level in a dose-dependent manner in OVCA432 cells. A significantly enhanced antitumor activity was observed with the combination of ExoPAC and ExoAnthos against A2780 tumor xenografts. Together, our data indicate that the berry Anthos are highly effective against ovarian cancer and that the milk exosomes serve as an excellent nano-carrier to enhance the drug's oral bioavailability for the management of ovarian cancer.


Introduction

Ovarian cancer is the most lethal gynecological malignancy in women in the United States.1 Five to 10 percent of ovarian cancers diagnosed is hereditary.2 Front-line treatment for this lethal disease is cytoreductive surgery followed by intraperitoneal (i.p.) and/or intravenous (i.v.) platinum chemotherapy.3,4 Within 5 years after the initial treatment, the disease recurs in 60–70% of patients4 and often exhibits cross-resistance to many structurally-related or unrelated drugs. In addition, ∼25% of ovarian cancers are “innately” resistant to platinum and respond poorly to initial chemotherapy.5 For the treatment of ovarian carcinomas, therefore, cisplatin resistance presents a very serious problem. Strategies tried to overcome such chemoresistance include combining platinum-based chemotherapy with new molecularly-targeted drugs and other chemotherapeutic drugs. Paclitaxel (PAC) is the most effective agent in such patients with relapsed platinum-refractory disease. Many of these drug combinations have severe toxic side effects and fail to increase the survival rates of patients with drug-resistant tumors.6 Therefore, there is an urgent need to develop effective treatment strategies for the management of platinum-resistant ovarian cancers. Therapeutic dosing with non-toxic plant bioactives in combination with chemotherapeutic drugs to treat the ovarian cancer and overcome drug resistance is a viable strategy.

Epidemiologic studies have shown constant inverse associations between the intake of dark-colored fruits and vegetables and incidence of several chronic diseases.7 Dark-colored fruits and vegetables have generally been accepted as cancer preventives. Berries have shown potential to prevent chemically-induced colon8 and esophageal9 cancers in animal models. Furthermore, we have shown the chemopreventive and therapeutic activities of a diet supplemented with freeze-dried berry powder against estrogen (E2)-mediated mammary cancer.10–15 More recently we demonstrated the significant therapeutic activity of dietary berries against a lung cancer xenograft.31 Translatability of dietary berries for therapeutic effects to a large population, however, is not practical due to rather large daily doses of berries. In order to achieve effective doses for clinical translation, we have used the putative berry bioactives, namely, anthocyanidins (Anthos) instead of the whole fruit. Berry bioactives possess potent anti-oxidant, anti-proliferative, apoptotic and anti-inflammatory properties.16,17,19,31 The berry phytochemicals have significant therapeutic activity against breast cancer in vivo.18 Our own published data demonstrate that Anthos have significant therapeutic activity against lung cancer in both cell culture and animal studies.19 Anthocyanins were shown to overcome trastuzumab-resistance to inhibit the breast cancer growth.20 However, it is well documented that berry bioactives exhibit poor oral bioavailability and stability thus limiting their effectiveness to full potential for clinical applications.21,22 Identifying effective oral delivery systems that can achieve high oral bioavailability and are safe when used for prolonged periods are needed to overcome the limitations of Anthos for clinical translatability.

Exosomes are endogenous nanoparticles secreted by a wide variety of cells and are explored as nano-carriers for drug delivery.23,24 Data from our recent studies25–27 indicate that milk exosomes may provide a viable alternative with a high impact because of their nanosize, a scalable source, biocompatibility, ability of exosomes to stabilize drugs, lack of toxicity, tumor targetability, etc. Due to their known stability in an acidic environment,26,28 milk exosomes may provide a desirable oral drug delivery carrier, with wide preventive and therapeutic applications.

In this study we demonstrate that the exosomal formulation of Anthos elicits potent therapeutic activity against both the drug-sensitive and drug-resistant human ovarian cancer cells, as well as synergistically enhances the therapeutic activity when used in combination with cisplatin. Furthermore, the combination of ExoAnthos and ExoPAC treatment sensitized cisplatin resistant ovarian cancer cells by decreasing the PgP expression levels.

Materials and methods

The native mixture of Anthos was isolated in the laboratory from 36% anthocyanin-enriched bilberry extract (Indena, Seattle, WA) using a recently developed solvent–solvent extraction procedure (Patent # 8987481 B1).29 The Anthos thus isolated, with over 85% purity, was generously provided by 3P Biotechnologies, Inc. (Louisville, KY) which was further purified by using a C18 Bond Pak column to ∼95% purity as determined by UPLC. The Anthos mixture contains glycosylated forms of delphinidin (Dp), cyanidin (Cy), malvidin (Mv), peonidin (Pe) and petunidin (Pt) in the proportion of 33[thin space (1/6-em)]:[thin space (1/6-em)]28[thin space (1/6-em)]:[thin space (1/6-em)]16[thin space (1/6-em)]:[thin space (1/6-em)]16[thin space (1/6-em)]:[thin space (1/6-em)]7 as described previously.19 The molecular weight of the Anthos mixture represents the average mol. wt of these five individual anthocyanidins calculated by multiplying their content with their respective molecular weights. Authentic Anthos standards were purchased from Chromadex (Irvine, CA).The primary p-glycoprotein antibody and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). All other chemicals received were of analytical grade.

Isolation of exosomes and their characterization

Exosomes were isolated from mature bovine milk by differential centrifugation as described previously.27 The isolated exosomes were characterized by the analysis of size and surface protein markers (CD63 and CD81, etc.). The protein markers were characterized by western blotting as described.27 The exosomes were suspended in PBS and stored at −80 °C until use. The size distribution of the isolated exosomes was measured by using a NanoSight and a Zetasizer. The size was confirmed by scanning electron microscopy (SEM) and atomic force microscopy (AFM) as described previously.27,30

Drug encapsulation

Drug loading was achieved by dissolving the test agents in ethanol and then mixing with the milk exosomes. The free drug was removed by a low-speed centrifugation (8000g, 5 min) and the drug-loaded exosomes were collected by either Amicon ultrafiltration using a 500 kDa filter (for ExoAnthos) or by ultracentrifugation (135[thin space (1/6-em)]000g, 90 min). The drug loading was determined by the analysis of the drug and protein in the formulation as described previously.27,30

Cell lines

Human ovarian cancer cells (A2780, A2780/CP70, OVCA432, and OVCA433) were kindly provided by Dr J. Christopher States, University of Louisville, Louisville, KY. All cells were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, 2 mM L-glutamine, and 0.2 units ml−1 insulin (Sigma-Aldrich, St Louis, MO). Cells were cultured under an atmosphere of 95% humidity and 5% CO2 at 37 °C.

Antiproliferative activity

The antiproliferative activity of the Anthos, PAC, cisplatin and the combination of these drugs and their exosomal formulations was assessed against ovarian cancer cells by using the MTT assay. All the cell lines were plated (5000 cells per well) and the antiproliferative activity was determined after 72 h as described earlier against lung cancer cells.27,30

Western blot analysis

The ovarian cancer OVCA432 cells were treated with the Anthos and PAC, individually and in combination at different concentrations. After 24 h cells were collected and whole cell lysates were prepared in RIPA buffer. The protein concentration was determined using the BCA method. Proteins were separated on the SDS-PAGE gel, and after that the transfer membranes were blocked with 4% non-fat dairy milk in TBS-T and incubated with the primary antibodies overnight at 4 °C. After washing, the membrane was probed with a secondary antibody and protein bands were visualized with chemiluminescence detection kits (Amersham, ECL kits, Sunnyvale, CA).

Ovarian cancer xenografts

All animal experiments were conducted in accordance with the applicable institutional and national guidelines and regulations for the care and use of animals in research. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Louisville prior to initiation. The UofL animal care and use program is fully accredited by AAALAC International. Female athymic nude mice (5–6-week old) were purchased from Harlan Laboratories (Indianapolis, IN), acclimated for a week and then inoculated with human ovarian cancer A2780 cells (1.5 × 106 cells) in Matrigel. Once tumor grew to around 80 mm3, animals were randomized into several groups (n = 8–10 in each group) and different studies were conducted as described below.

In study 1, animals were treated via oral gavage with a vehicle, two doses of Anthos (6 and 30 mg kg−1 b. wt), and the exosomal formulation of Anthos containing 6 mg kg−1 Anthos and 60 mg kg−1 Exo three times a week. In study 2, animals were treated with the chemotherapeutic drug, PAC (4 mg kg−1 b. wt), and the exosomal formulation of PAC (4 mg PAC kg−1 b. wt and 60 mg Exo kg−1 b. wt) given i.p. and by oral gavage, respectively. Additional groups were treated with ExoAnthos and a combination of ExoAnthos and ExoPAC at the doses described above.

The animal weight, diet intake and tumor growth were monitored weekly. Due to the highly aggressive nature of the A2780 cells, all studies were terminated after three weeks by euthanizing animals by CO2 asphyxiation.

Statistical analysis

The results are presented as the average and standard error or standard deviation of three experiments done in triplicate. Statistical analysis was performed using the GraphPad Prism statistical software. Student's t-test was performed for the statistical analysis and a p-value of <0.05 was considered significant. Data were further analyzed for statistical significance using the analysis of variance (one way ANOVA).

Results and discussion

Antiproliferative activity of Anthos

The cisplatin-resistant (A2780/CP70, OVCA432 and OVCA433) cells overexpress PgP and show 100-fold resistance to cisplatin compared to the drug-sensitive (A2780) ovarian cancer cell lines (Fig. 1A and C). We examined the antiproliferative activity of Anthos against drug-sensitive and cisplatin-resistant ovarian cancer cells. Anthos resulted in a dose-dependent inhibition of ovarian cancer cells (Fig. 1B). Anthos demonstrated an IC50 value of nearly 25 μM against the sensitive A2780 cells; however, it was almost 10-fold higher for the drug-resistant OVCA432 cells (Fig. 1C). This is the first demonstration showing the ability of the Anthos to inhibit the growth of drug-resistant ovarian cancer cells. We have shown the antiproliferative and anti-cancer activities of the Anthos against lung cancer cells and demonstrated that the Anthos presumably target distinct and overlapping molecular signaling pathways resulting in greater anti-cancer effects than the individual moieties.19
image file: c7fo00882a-f1.tif
Fig. 1 Antiproliferative activity of the chemotherapeutic drug, cisplatin (a) and the native mixture of Anthos from bilberry (b) against drug-sensitive (A2780) and drug-resistant (A2780/CP70, OVCA432 and OVCA433) human ovarian cancer cells by using the MTT assay. (c) Shows IC50 values calculated by using CalcuSyn software. Standard deviation (5–7%) is not shown for clarity. IC50 values followed by different letters are significantly different at p ≤ 0.05.

ExoAnthos enhance growth inhibition

The relative antiproliferative effects of ExoAnthos and Anthos were determined against cisplatin-resistant human ovarian cancer OVCA433 cells by using the MTT assay. ExoAnthos showed a significant reduction in the drug concentration to achieve growth inhibition versus the free Anthos (Fig. 2). A 20-fold difference in the IC50 value was observed between ExoAnthos and Anthos. We reported similar findings with another cisplatin-resistant ovarian cell line (OVCA432) where the Anthos exhibited an IC50 value of 112 μM which was decreased to 7 μM with the ExoAnthos.25
image file: c7fo00882a-f2.tif
Fig. 2 Anti-proliferative activity of Anthos-loaded milk exosomes vs. free Anthos against human drug-resistant ovarian cancer OVCA433 cells by using the MTT assay. The exosomal protein concentration was kept constant (50 μg ml−1). Each data point represents the average of 3–4 replicates ± sd. Students’ t-test was conducted to calculate the statistical significance. **p < 0.01, ***p < 0.001.

The enhanced activity of the ExoAnthos could be presumably due to the stability of the Anthos after encapsulation into/onto exosomes.25 In addition, greater inhibition could also be due to the higher uptake of the Anthos in the exosomal formulation. We have previously shown that exosomes are taken up by the cancer cells relatively rapidly and can achieve the maximum level in 4–8 h.27 Interestingly, the exosomes per se resulted in moderate (about 18%) growth inhibition of the OVCA433 cancer cells. These findings indicate that the milk exosomes play an important role in enhancing the efficacy of drugs.

Anti-tumor activity of Anthos and ExoAnthos

Our previous studies have indicated the therapeutic activities of dietary berries and Anthos against breast and lung cancers.11,19,31 To determine the therapeutic potential of Anthos against ovarian cancer, female athymic nude mice were subcutaneously inoculated with the A2780 ovarian cancer cells (1.5 × 106 cells) and when the tumor xenograft reached about 80 mm3, animals were treated with the Anthos at 30 mg kg−1 b. wt by oral gavage. The data showed an inhibition of about 63% with the Anthos treatment compared with the vehicle control (575 ± 217 vs. 1535 ± 340) (Fig. 3A). Anthos at a dose of 20 mg kg−1 (i.p.) has been shown to significantly inhibit the lung tumor growth in this model.19 A higher dose (30 mg kg−1 b. wt) was chosen because of the aggressive nature of ovarian cancer (A2780) cells and the mode of delivery (i.p. vs. oral gavage). Nevertheless, these studies are the first to show that the Anthos are capable of inhibiting ovarian cancer.
image file: c7fo00882a-f3.tif
Fig. 3 Antitumor activity of the Anthos (6 and 30 mg kg−1 b. wt) and the exosomal formulations of Anthos (6 mg kg−1 b. wt, 60 mg kg−1 Exo protein) given by oral gavage and compared with the vehicle control against the ovarian cancer A2780 tumor xenograft. Sub-optimal concentrations of Anthos (6 mg kg−1) were used to appreciate the effect of the exosomal formulation. *p < 0.05.

To determine if the exosomal formulation of the Anthos exhibit a higher anti-tumor activity than the free Anthos, we subcutaneously inoculated nude mice with the A2780 cells. When the tumors grew to >80 mm3, animals were treated orally with a vehicle, sub-optimal doses of the Anthos (6 mg kg−1) and ExoAnthos (6 mg Anthos and 60 mg Exo protein kg−1 b. wt), three times a week. We opted to use a suboptimal dose of the Anthos in order to appreciate the anticipated enhanced efficacy of the exosomal formulation. The results indicated that the exosomal formulation even at a sub-optimal dose of the Anthos significantly inhibited the tumor growth (p < 0.05); the free Anthos showed a slight but insignificant growth inhibition at this low dose (Fig. 3B). The data also provide support to the notation that Anthos are protected in the exosomal formulation and remain viable after tolerating the GI conditions to elicit a biological response. We have previously reported nearly 20% tumor growth inhibition by the milk exosomes alone against the lung cancer xenograft.27,30 The higher efficacy of ExoAnthos in vivo suggests that Exo formulations possibly provide greater stability, higher uptake, and longer circulation time and slow release of the Anthos from the exosomes to boost the anticancer effects. However, these parameters need to be exhaustively studied under in vivo conditions to draw definitive conclusions regarding how exosomes increase the efficacy of drugs.

Anthos enhance the therapeutic response of cisplatin

In clinical scenario drug-resistant ovarian tumors are often treated by combining platinum-based chemotherapy with other chemotherapeutic drugs to overcome chemoresistance. We examined if the Anthos can chemosensitize the drug-resistant OVCA432 cells to cisplatin. The data in Fig. 4A demonstrate a significantly higher cell killing (10–15 fold lower IC50) of cisplatin-resistant OVCA432 cells by cisplatin when combined with 75 μM Anthos. These results suggest that the Anthos have potential to chemosensitize the drug-resistant ovarian cancer cells and reduce the effective cisplatin dose required to achieve the therapeutic response.
image file: c7fo00882a-f4.tif
Fig. 4 Cell viability of the drug-resistant human ovarian cancer (OVCA433) cells treated with cisplatin alone or in combination with 75 μm Anthos (a). Panel b shows the effect of Anthos and PAC on the PgP expression. Cells were treated with the indicated concentrations of PAC, Anthos and a combination of the two for 24 h. Cells were collected and proteins were separated on SDS-PAGE, and probed for the p-glycoprotein. β-Actin was used as the loading control. *p < 0.05, **p < 0.01, ***p < 0.001.

Anthos inhibit the PgP expression in the presence of PAC

PAC is used to treat drug-resistant recurrent ovarian cancers as they often retain sensitivity to PAC.6 We examined if PAC and Anthos affect the PgP expression levels in order to overcome drug resistance in ovarian cancer cells. Drug-resistant OVCA432 cells were treated with the Anthos and PAC, alone and in combination, and whole cell lysates were analyzed by western blotting. The findings show that the Anthos and PAC alone do not have a significant effect on the expression of PgP. However, the PgP expression was significantly reduced (∼30%) in the cells treated with the combination regimen (Fig. 4B). Our data corroborate a recent report in which Anthos have been shown to inhibit PgP as analyzed by using the calcein extrusion assay.32 The inhibition of PgP represents a promising therapeutic strategy for ovarian cancer patients. These data provide preliminary evidence suggesting the potential benefit of combining PAC and Anthos to treat drug-resistant ovarian tumors.

Anthos enhance the therapeutic activity of PAC in vivo

We further evaluated whether the Anthos could enhance the sensitivity of human ovarian cancer xenografts to the chemotherapeutic drug, PAC in vivo. Our previous findings indicated that the ExoPAC formulation given orally resulted in a significantly higher anti-tumor efficacy compared to PAC administered i.p at a 4 mg kg−1 b. wt dose against the lung cancer xenografts.26 In addition, the ExoPAC formulation given orally was found to substantially reduce the PAC-associated toxicities in wild-type mice.26 In view of these findings we decided to test the ExoPAC formulation instead of free PAC in combination with the Anthos against drug-resistant OVCA432 and drug-sensitive A2780 ovarian tumor xenografts.

Due to unknown reasons, the inoculation of 10 × 106 OVCA432 cells into the right flank of athymic nude mice did not produce any measurable tumors as described by Takai and colleagues.33 It is possible that OVC432 tumors require exogenous estrogen in view of their estrogen dependency in the cell culture system. We have shown the slow release of estrogen via silastic implants,34 this remains to be tested with OVCA tumor xenografts. While the inoculation of 1.5 × 106 A2780 cells produced tumors measuring approximately 80 mm3 after 1 week of cell inoculation. Therefore, the A2780 tumor xenografts were treated with the combination of ExoAnthos and ExoPAC. Tumor-bearing animals received ExoAnthos (6 mg kg−1, 60 Exo protein) on alternate days, ExoPAC (4 mg kg−1, 60 mg kg−1 Exo protein) weekly, or a combination of ExoAnthos and ExoPAC. The study was terminated after 2.5 weeks. The results indicate significant tumor growth inhibition with ExoAnthos (65%) and the combination (78%) treatment (Fig. 5), while ExoPAC showed only modest but insignificant tumor inhibition. This lack of tumor inhibition with ExoPAC is in contrast to the significant inhibition of the A549 lung tumor xenograft at the same oral dose of ExoPAC in our previous study,26 and may presumably require a higher dose of ExoPAC considering the aggressive nature of the A2780 ovarian cancer cells. Nevertheless, these findings confirm the synergistic effects of the ExoAnthos and ExoPAC combination to treat ovarian tumors.


image file: c7fo00882a-f5.tif
Fig. 5 Effect of the exosomal formulations on the ovarian cancer (A2780) tumor xenograft. Once tumor grew to about 80 mm3, animals were treated with a vehicle, PAC alone, ExoPAC (4 mg kg−1), ExoAnthos (6 mg kg−1) and a combination of the two by oral gavage on alternate days. *p < 0.05, **p < 0.01.

Conclusion

Ovarian cancers are difficult to treat due to the development of chemoresistance. Hence, new strategies are needed for the effective management of ovarian cancers. We demonstrate that the Anthos can be used to sensitize drug-resistant ovarian cancer cells. Notably, the Anthos reduced the effective dose of cisplatin required to inhibit cisplatin-resistant ovarian cancer cells. These anti-tumor effects are enhanced by the exosomal formulation of the Anthos presumably due to increased stability and bioavailability compared to the free Anthos. Further combination of the Anthos and PAC decreased the PgP expression to sensitize cisplatin-resistant ovarian cancer cells. In summary, our data indicate that the exosomal formulation of the Anthos has potential to sensitize resistant tumors and enhance the therapeutic efficacy of chemotherapeutic drugs used in the management of ovarian cancers.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the Agnes Brown Duggan Endowment and Helmsley Trust Funds. R. C. G. holds the Agnes Brown Duggan Chair in Oncological Research. We also acknowledge Dr J. Christopher States, Professor, Department of Pharmacology and Toxicology, University of Louisville for kindly providing the ovarian cancer cell lines used in this study and 3P Biotechnologies, Inc. (Louisville, KY) for providing bilberry-derived anthocyanidins.

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