Design and application of hybrid cyclic-linear peptide-doxorubicin conjugates as a strategy to overcome doxorubicin resistance and toxicity

https://doi.org/10.1016/j.ejmech.2021.113836Get rights and content

Highlights

  • [R5K]W7A-Dox conjugate had 16-fold higher activity against Dox-resistant cells versus Dox.

  • The conjugate significantly reduced the cell cytotoxicity in heart cells versus Dox.

  • The conjugate exhibited comparable antiproliferative activity to free Dox in cancer cells.

  • This conjugate can be used as a potential anticancer prodrug.

Abstract

Doxorubicin (Dox) is used for breast cancer, leukemia, and lymphoma treatment as an effective chemotherapeutic agent. However, Dox use is restricted due to inherent and acquired resistance and an 8-fold increase in the risk of potentially fatal cardiotoxicity. Hybrid cyclic-linear peptide [R5K]W7A and linear peptide R5KW7A were conjugated with Dox through a glutarate linker to afford [R5K]W7A-Dox and R5KW7A-Dox conjugates to generate Dox derivatives. Alternatively, [R5K]W7C was conjugated with Dox via a disulfide linker to generate [R5K]W7C–S–S-Dox conjugate, where S–S is a disulfide bond. Comparative antiproliferative assays between conjugates [R5K]W7A-Dox, [R5K]W7C–S–S-Dox, linear R5KW7A-Dox, the corresponding physical mixtures of the peptides, and Dox were performed in normal and cancer cells. [R5K]W7A-Dox conjugate was 2-fold more efficient than R5KW7A-Dox, and [R5K]W7C–S–S-Dox conjugates in inhibiting the cell proliferation of human leukemia cells (CCRF-CEM). Therefore, hybrid cyclic-linear [R5K]W7A-Dox conjugate was selected for further studies and inhibited the cell viability of CCRF-CEM (84%), ovarian adenocarcinoma (SK-OV-3, 39%), and gastric carcinoma (AGS, 73%) at a concentration of 5 μM after 72 h of incubation, which was comparable to Dox (5 μM) efficacy (CCRF-CEM (85%), SK-OV-3 (33%), and AGS (87%)). While [R5K]W7A-Dox had a significant effect on the viability of cancer cells, it exhibited minimal cytotoxicity to normal kidney (LLC-PK1, 5–7%) and heart cells (H9C2, <9%) at concentrations of 5–10 μM (compared to free Dox at 5 μM that reduced the viability of kidney and heart cells by 85% and 44%, respectively). The fluorescence microscopy images were consistent with the cytotoxicity studies, indicating minimal uptake of the cyclic-linear [R5K]W7A-Dox (5 μM) in H9C2 cells. In comparison, Dox (5 μM) showed significant uptake, reduced cell viability, and changed the morphology of the cells after 24 h. [R5K]W7A-Dox showed 16-fold and 9.5-fold higher activity against Dox-resistant cells MDA231R and MES-SA/MX2 (lethal dose for 50% cell death or LC50 of 2.3 and 4.3 μM, respectively) compared to free Dox (LC50 of 36–41 μM, respectively). These data, along with the results obtained from the cell viability tests, indicate comparable efficiency of [R5K]W7A-Dox to free Dox in leukemia, ovarian, and gastric cancer cells, significantly reduced toxicity in normal kidney LLC-PK1 and heart H9C2 cells, and significantly higher efficiency in Dox-resistant cells. A number of endocytosis inhibitors did not affect the cellular uptake of [R5K]W7A-Dox.

Introduction

Doxorubicin (Dox) is a well-known, widely used anthracycline anticancer agent and has been approved by the FDA for the treatment of leukemias, sarcomas, and lymphoma, as well as breast, gastric, ovarian, lung, and thyroid cancer [1]. Dox acts through the inhibition of topoisomerase II (TOPO II)−DNA complex. DNA damage occurs by intercalating of Dox with the DNA double helix [2,3].

The inherent resistance in cells that have never been exposed to anticancer drugs and the development of resistance after the initial response is one of the major limitations of cancer chemotherapy treatment. Several mechanisms of inherent and acquired multi-drug resistance have been studied, including alteration of the target protein and drug metabolism, decreased membrane permeability, and/or efflux pumping [[4], [5], [6]]. Dox use for treating some types of tumors, e.g., ovarian carcinoma, liver cancer, and stomach cancer, has become complicated due to the development of resistance associated with it [7,8].

Moreover, the clinal application of Dox has demonstrated undesirable pharmacokinetic properties, such as low bioavailability, rapid distribution, and excretion of the drug, due to the hydrophilic nature and a high volume of distribution [[9], [10], [11], [12]]. Thus, a higher dose of Dox is required in cancer chemotherapy to achieve an adequate therapeutic effect. However, higher cumulative dose leads to dose-dependent side effects, such as cardiotoxicity, nephrotoxicity, and extravasation [13,14].

Intracellular Dox accumulation is dependent on many parameters, such as cellular uptake, retention, re-localization, and efflux from the cells. Among these factors, uptake of Dox is affected by the efflux mechanism in several cancer cells such as ovarian carcinoma cells that leads to the decreased levels of intracellular Dox. The overexpression of energy-dependent efflux pump integral membrane proteins such as P-glycoprotein (P-gp) removes drugs and thus reduces intracellular anticancer drug concentrations [15].

The biological efficacy and toxicity of an anticancer drug can be modified by using drug delivery systems and altering the physicochemical properties, such as lipophilicity, cellular uptake, and prolonging activity through chemical conjugation with various chemical moieties. One of the main applications of drug delivery systems is avoiding the P-gp and multi-drug resistance proteins (MRPs) involved in drug efflux to overcome the resistance problem and P-gp-mediated drug efflux [[16], [17], [18]].

Chemical conjugation with a parent drug has been widely used as one of the drug delivery systems, which is referred to as a prodrug strategy [[19], [20], [21]]. Several methods have been used to improve Dox delivery, including using gold nanoparticles [22], gold nanospheres [23], liposomes [24], peptides [[25], [26], [27], [28], [29]], and dendrimers [30]. There are many other reported studies on conjugated-based nanomedicines for cancer therapy [[31], [32], [33]]. We have previously reviewed several delivery systems of Dox, including metal nanoparticles, carbon nanotubes, dendrimers, liposomes, fullerenes, cyclic peptides, and other covalent/non-covalent systems [34].

Chemical conjugation of peptides with a parent drug has been widely used in Dox delivery [21,35]. Conjugation of Dox with cell-penetrating peptides (CPPs) has been employed as one of the most effective methods to translocate the drug into various cell lines. For instance, Dox has been conjugated with different linear CPPs, including penetratin [26], TAT [36], polyarginine [37], and maurocalcine [38]. However, many of the above internalization mechanisms of CPP-based delivery systems involve endocytosis. Furthermore, many of these delivery systems did not resolve the cardiotoxicity of Dox. Finally, it is still challenging to develop efficient and safe prodrug carriers to enhance the delivery and retention of Dox into drug-resistant tumor cells. Thus, it gave us the impetus to design and synthesize more variations of peptide structures to improve delivery and reduce the toxicity in normal cells.

Application of a number of cyclic CPPs as noncovalent nuclear targeting molecular transporters of Dox has also previously been reported [39]. Cyclic peptides containing arginine (R) and tryptophan (W), [WR]n (n = 4–5), were found to be appropriate noncovalent carriers for Dox. We have previously reported the conjugation of cyclic CPPs such as [WR]5 containingW and R residues with enhanced intracellular delivery of paclitaxel (PTX), camptothecin (CPT) [40], curcumin [41], and Dox [42,43] in different cancer cell lines escaping endosomal pathways. For example, the conjugation of Dox with a cyclic CPP, cyclic [W(RW)4]-Dox, improved the antiproliferative activity of Dox compared to the corresponding linear (RW)4−Dox in human leukemia (CCRF-CEM), ovarian adenocarcinoma (SK-OV-3), colorectal carcinoma (HCT-116), and breast carcinoma (MDA-MB-468). The cyclic peptide-Dox conjugate showed comparable antiproliferative activity at a concentration of 1 μM against CCRF-CEM and MDA-MB-468 when compared to Dox [42]. [W(RW)4]−Dox, significantly improved the cellular uptake and retention time of Dox in SK-OV-3 cancer cells. Flow cytometry analysis showed 3.3–3.6-fold higher cellular uptake of cyclic [W(RW)4]−Dox than Dox alone and the physical mixtures, cyclic [W(RW)4] + Dox and linear (RW)4 + Dox, in SK-OV-3 cells after 24 h incubation [42].

We have also synthesized and evaluated the activity of Dox thiol conjugates, thiolated doxorubicin (Dox-SH), thiol-reactive Dox-S-S-Pyr, and a Dox-S-S-cell-penetrating cyclic peptide, Dox-S-S-[C(WR)4K], and compared their cytotoxicity to Dox alone. All compounds showed high cellular uptake and were localized mostly in the nucleus in various cell lines [43]. Our study further demonstrated that Dox-S-S-[C(WR)4K] exhibited less cytotoxicity in mouse myoblast cells in comparison to Dox [43].

Our previous studies have demonstrated that hybrid cyclic-linear peptides [R5K]W6 and [R5K]W5 exhibited high efficiency to deliver siRNA into the MDA-MB-231 and MDA-MB-468 cells [44]. Recently, we discovered that hybrid cyclic-linear peptide [R5K]W7 significantly improved the uptake of a fluorescence-labeled phosphopeptide (F'-GpYEEI), emtricitabine (F'-FTC), and stavudine (F'-d4T) (F′ = 5(6)-carboxyfluorescein) by approximately 48, 27, and 36 folds, respectively, when compared with parent fluorescence-labeled compounds alone in CCRF-CEM cells after 3 h incubation (data not shown here). Thus, herein we report the synthesis of linear R5KW7A (1), cyclic-linear [R5K]W7A (2), and cyclic-linear [R5K]W7C (3) peptides (Fig. 1). The peptides were used for the synthesis of peptide-Dox conjugates (4–6, Fig. 2) through an ester or disulfide linkage. The antiproliferative activities of the peptide-Dox conjugates were evaluated in multiple cancer, normal, and Dox-resistant cell lines. The linear and cyclic-linear peptides were selected for comparative studies. The mechanism of cellular uptake was investigated in the presence of endocytosis inhibitors. To the best of our knowledge, this is the first report of hybrid cyclic-linear peptide-Dox conjugates to circumvent Dox resistance and cardiotoxicity.

Section snippets

Materials

All protected amino acids and resins were purchased from AAPPTEC (Louisville, KY, USA). Doxorubicin was purchased from LC Laboratories (Woburn, MA, USA). All the other chemicals reagents were purchased from MilliporeSigma (Milwaukee, WI, USA). Medium (RPMI-1640), fetal bovine serum, and all other cell biology reagents were purchased from Wilkem Scientific (Pawtucket, RI, USA) and Fisher Scientific (Hanover Park, IL, USA). 4′,6′-Diamidino-2-phenylindole (DAPI) was purchased from Vector

Chemistry

Linear and hybrid cyclic-linear peptides were synthesized by Fmoc/tBu solid-phase peptide synthesis. The linear peptide R5KW7A was assembled on the H-Arg(Pbf)-2-chlorotrityl resin. The resin was dried, washed, and cleaved by a cleavage cocktail (reagent R) to afford the linear R5KW7A (1) (Fig. 1), which was purified by reversed phase HPLC. For the synthesis of the hybrid cyclic-linear peptides [R5K]W7A (2) and [R5K]W7C (3) (Fig. 1), the linear protected peptides, R5K(Dde)W7A and R5K(Dde)W7C

Discussion

Dox is a widely used potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity [58]. Dox is one of the effective chemotherapeutic agents used for breast cancer, leukemia, and lymphoma treatment [59]. It has been approved by FDA for the treatment of leukemias, sarcomas, and lymphoma, as well as breast, gastric, ovarian, lung, and thyroid cancer. However, Dox also exerts non-specific toxicity in major organs and is particularly known

Conclusions

In summary, linear and cyclic-linear peptide-Dox conjugates were synthesized as prodrugs and were evaluated for their activities against various cancer cell lines in comparison to the free drug and the corresponding physical mixtures. [R5K]W7A-Dox conjugate demonstrates better antiproliferative activity when compared with [R5K]W7C–S–S-Dox and linear R5KW7A-Dox in CCRF-CEM cells. [R5K]W7A-Dox, exhibited comparable antiproliferative activity to free Dox after 72 h incubation time in all cancer

Author contributions

K.P., R.K.T. and H.M.A. planned and designed the experiments; S.M., P.H., and D.S. performed the chemistry; S.M., P.M., and D.S. conducted the cell-based assays; K.P., H.M.A. and R.K.T. contributed reagents/materials/analysis tools; K.P., S.M., H.M.A., and R.K.T. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Chapman University School of Pharmacy.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors also acknowledge the support of the core facility at Chapman University School of Pharmacy.

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