PEG含有高分子ナノキャリアとの4-チアゾリジンベースの化学療法剤の水分散複合体の増強されたアポトーシス促進効果

結果

遊離型および高分子ナノキャリアとの複合体で使用される4-チアゾリジノン誘導体の細胞毒性効果（トリパンブルー排除試験）

トリパンブルー排除試験の結果は、PNCとのLes-3288およびLes-3833複合体の水ベースの形態が、神経膠腫C6細胞（24時間および48時間の治療）に対する毒性が、これらの化合物の遊離型（図5および6）。最高の細胞毒性効果は、PNCとの複合体中のLes-3288およびLes-3833化合物の0.1および0.5μM用量で観察されましたが、これらの化合物の最高用量（1.0μM）では、PNCとの複合体形成の増強が検出されましたLes-3288の場合のみ24時間（図5および6）。

ポリマーナノキャリア（ PNC） * P <0.05; ** P <0.01; *** P <0.001 （ コントロールとの違い、100％）

ポリマーナノキャリア（ PNC） * P <0.05; ** P <0.01; *** P <0.001（コントロールとの差、100％）

Les-3833およびLes-3288化合物と高分子ナノキャリアの複合体形成は、ラット神経膠腫C6細胞に対するinvitroでのアポトーシス促進活性を増強します

選択した4-チアゾリジノン誘導体の細胞毒性の可能性に対するPNCの調節効果を評価するために、2つの代替アプローチが使用されました。最初に、細胞周期分析を実施して、細胞周期に対する薬物-PNC複合体の影響を調査した。さらに、アネキシンV / PI二重染色を利用して、これらのナノコンポジットによって誘発された細胞死の正確なタイプを区別しました。

低用量（0.1μg/ mLおよび0.5μg/ mL）のLes-3288およびLes-3833化合物は、図7および表1に示すように、細胞周期の進行またはアポトーシスの誘導のいずれにも目に見える影響を与えませんでした。 PNCを含む両方の化合物は、pre-G ₁ の細胞数を大幅に増加させました。 フェーズ（Les-3288の場合は4倍、Les-3833の場合は6倍）。これは、アポトーシス促進能の大幅な向上を示しています。遊離型のPNCは細胞周期やアポトーシス誘導に影響を与えなかったことを強調しておく必要があります。したがって、チアゾリジノン-ナノキャリア複合体で治療中のプレG1細胞の大集団の観察された出現は、PNCと実験薬の単純な相乗効果によって説明することはできません。

C6ラット神経膠腫細胞における遊離型および高分子ナノキャリア（PNC）との複合体における4-チアゾリジノン誘導体Les-3288およびLes-3833の細胞周期進行への影響。ヨウ化プロピジウム（PI）染色、フローサイトメトリー。 R2、G1以前; R3、G1; R4、S; R5、G2期。 FL2-H、フローサイトメーターの2番目のチャネル（585/40フィルター）のピーク発光値

観察されたサブG1ピークがアポトーシス細胞で構成されていることを確認するために、アネキシンV / PI染色アッセイの結果を分析しました。低用量（0.1および0.5μg/ mL）および高用量のLes-3288（1.0μg/ mL）でのC6神経膠腫治療中に、ホスファチジルセリン（初期アポトーシスの主なマーカー、FITC結合アネキシンVによって検出）の有意な外在化は観察されませんでした。 、この化合物がアポトーシスの弱い誘導物質であることを示唆している。代わりに、壊死細胞集団の増加（コントロールの1.2％からLes-3288の11.25％、1.0μg/ mL）が見つかりました。これは、Les-3288が少なくとも部分的に壊死細胞死を引き起こすことを示している可能性があります。しかし、Les-3288とPNCの複合体形成は、この薬剤の作用機序を完全に変えました。初期アポトーシス細胞（アネキシンV（+）/ PI（-））と後期アポトーシス細胞（アネキシンV（+）/ PI（+））の数が大幅に増加しましたが、純粋な壊死細胞の総量はコントロール（未処理）細胞のレベル（図8a）。したがって、Les-3288をPNCと結合させると、神経膠腫細胞に対するアポトーシス促進活性が強力に増強されるだけでなく、壊死促進性細胞死の潜在的なメカニズムも低下します。これらの所見は、患者にとって不便な炎症の大規模な誘発のために壊死促進薬の使用が推奨されていないため、臨床診療にとって非常に重要である可能性があります。

Les-3288のアポトーシス促進活性の比較（ a ）およびLes-3833（ b ）ラット神経膠腫C6細胞に対する化合物および高分子ナノキャリア（PNC）とのそれらの複合体。アネキシンV / PI二重染色、フローサイトメトリー。 PI、ヨウ化プロピジウム

驚いたことに、Les-3833 + PNC複合体では同じ効果は観察されませんでした。実際、Les-3833 + PNC複合体のアポトーシス促進活性は、試験したすべての濃度で、遊離型のLes-3288と比較して低かった（図8b）。両方の化合物間のこのような劇的な違いは、それらの化学構造の特異性によって説明される可能性があり、Les-3833の場合、より良い結果を達成するためにポリマーの構造を最適化する必要があると推測されます。

遊離型でPNCと複合体を形成した4-チアゾリジノン誘導体によって引き起こされたラット神経膠腫C6細胞のDNA損傷のコメットアッセイ

アルカリコメットアッセイにより、アルカリに不安定なDNA部位での一本鎖DNA切断を検出できます。得られた結果は、オリーブテールモーメント（OTM）の平均値を使用して推定されました。私たちのデータは、Les-3288、Les-3833、およびPNC（濃度0.5μg/ mL）でのС6ラット神経膠腫細胞の3時間の処理（図9および10）は、重大なDNA損傷を引き起こさなかったことを示しました（OTM =0.7299± 0.1276、OTM =1.466±0.3086、OTM =0.5846±0.1078）、コントロールの未処理細胞と比較（OTM =0.4541±0.07273）。ただし、Les-3833 + PNC複合体（OTM =2.3880±0.2212）を使用した細胞処理は、遊離型のLes-3833を使用した細胞処理よりも重大なDNA損傷を引き起こしました。このような損傷の増加は、Les-3288 + PNC複合体の作用では観察されませんでした（図9および10）。

DNA損傷は、0.5μg/ mLの濃度で使用された実験的抗腫瘍性化合物で3時間処理されたラット神経膠腫C6細胞のオリーブテールモーメントを使用して評価されました。 * P ≤0.05; ** P ≤0.01; *** P ≤0.001。 PNC、高分子ナノキャリア;ドキソルビシン、ドキソルビシン（ポジティブコントロール）

（ a で3時間処理した後のラット神経膠腫С6細胞の尾の彗星におけるDNAの存在 ）コントロール（未処理の細胞）、Les-3288（ b ）、Les-3288 + PNC（ c ）、Les-3833（ d ）Les-3833 + PNC（ e ）、PNC（ f ）およびドキソルビシン（ g ), used at a 0.5 μg/mL concentration

An increase of cell incubation time to 6 h (Figs. 11 and 12) caused greater DNA damage in all experimental groups:Les-3288, Les-3288+PNC, Les-3833, Les-3833+PNC, and Dox (positive control). However, the effects of the Les-3288+PNC and Les-3833+PNC complexes appeared to be lower than the effects of the free forms of Les-3288 and Les-3833. It should be noted that the DNA-damaging effect of the complexes of both derivatives with the PNC was less than such effect from these derivatives used in free form.

DNA damage was evaluated using the Olive tail moment in rat glioma C6 cells treated for 6 h with experimental antineoplastic compounds used at a 0.5 μg/mL concentration. * P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. PNC, polymeric nanocarrier; Dox, doxorubicin (positive control)

Presence of DNA in comet tail of rat glioma С6 cells after treatment for 6 h with (a ) control (untreated cells), Les-3288 (b ), Les-3288 + PNC (c ), Les-3833 (d ) Les-3833 + PNC (e ), PNC (f ), and doxorubicin (g ), used at a 0.5 μg/mL concentration

The results were evaluated using the mean value of the TailDNA%. The obtained data show that incubation of С6 rat glioblastoma cells with substance Les-3288, Les-3288+PNC, Les-3833, and PNC (all at concentration of 0.5 μg/mL) during 3 h (Fig. 13) does not lead to significant DNA damage (TailDNA% of 4.157 ± 0.682; TailDNA% of 3.530 ± 1,012, 8.807 ± 0.878; 3.298 ± 0.514, respectively) compared with control (TailDNA% =3.638 ± 0.406), but cell treatment with the Les-3833+PNC complex (TailDNA% =19.53 ± 1.48) causes more significant damage of DNA than the free form of Les-3833. An increase in the incubation time up to 6 h (Fig. 14) in all cases leads to greater damage to the DNA. Again, the level of TailDNA% detected from the action of complexes of both derivatives with the PNC was less than the level of that indicator for the action of these derivatives used in free form.

DNA damage evaluated using the % of DNA in comet tail of C6 rat glioma cells treated with experimental antineoplastic compounds during 3 h at a concentration of 0.5 μg/mL. * P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. PNC, polymeric nanocarrier; Dox, doxorubicin (positive control)

DNA damage evaluated using the % of DNA in comet tail of C6 rat glioma cells treated with experimental antineoplastic compounds during 6 h at a concentration of 0.5 μg/mL. * P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. PNC, polymeric nanocarrier; Dox, doxorubicin (positive control)

We also used an alternative, five-category classification system to classify DNA migration and calculated the DI. Table 2 summarizes the percentages of rat glioma C6 cells for each level of genotoxicity during 3 h of treatment. In the control (untreated cells), higher percentages of cells in levels 0 (81.3%) and 1 (18.6%) were detected compared to these levels in Les-3288 and PNC-treated cells. Complexation of Les-3288 with the PNC did not lead to a big change in the distribution pattern of the DNA comets. In contrast, Les-3833-treated cells showed a lower percentage of cells in 0 level (38.6%) and a higher percentage of cells in level 1 (60.0%), while Les-3833 + PNC-treated cells demonstrated a significantly lower percentage of cells in level 0 (8.0%) and much higher percentages of cells with levels 1 (76.0%), 2 (13.3%), and 3 (2.6%). The integrative indicator of DNA damage (DI) in the case of application of both Les-3288 and its complex with the PNC did not differ significantly from that for control or using free PNC, while the DI from using both Les-3833 and its complex with the PNC was even higher than the DI from using Dox.

During this time interval, Les-3833 + PNC-treated cells demonstrated a higher percentage of cells with level 4 (5.3%) DNA damage than cells treated with Les-3288 and the other experimental compounds, but the percentages of cells in levels 2 and 3 were lower (16.0% and 12.0%, respectively) than those from treatment with other compounds (Table 2). Table 3 summarizes the percentages of rat glioma C6 cells with DNA damage after 6 h of treatment. In general, a tendency for the appearance of DNA comets of higher classes was detected when complexes of Les-3833 and Les-3288 were used, compared with a spectrum of DNA comet classes induced by free forms of these agents (Table 3). However, the integrative indicator of DNA damage (DI) calculated for the action of complexes of Les-3833 and Les-3288 was found to be lower than such an indicator for the action of free forms of these agents. A possible explanation could be a very high DI level at 6 h action of studied agents that is even higher than such indicator found for the action of Dox (Table 3). Thus, we observed time dependence of DNA damage caused by complexes of Les-3833 and Les-3288:at 3 h of treatment, there was an increase of the DI in the case of action of Les-3833 complexes, compared to the action of the free form of Les-3833; however, at 6 h of treatment, there was a decrease in DNA damage for the action of complexes of both Les-3833 and Les-3288, compared to such damage observed for the action of the free forms of these agents. There was no significant time dependence for the action of the PNC, while a significant time-dependent (from 3 to 6 h) increase in the DI was observed for the action of Dox (from 41.2 + 0.40 to 185.2 + 0.23).

DNA Intercalation by 4-Thiazolidinone Derivatives—Les-3288 and Les-3833

Les-3288 and Les-3833 compounds demonstrated a certain capability for intercalating DNA molecules with approximately 15% and 10%, respectively, of replacement of methyl green dye. EtBr, a well-known DNA intercalating agent, was used as a positive control, and it was shown to replace the methyl green dye much more efficiently (70%) (Fig. 15). Thus, Les-3288 and Les-3833 are not capable of effectively intercalating in between two complementary base pairs in double-stranded DNA.

Replacement of methyl green dye intercalated into the DNA of salmon sperm by Les-3288 and Les-3833 compounds (1.0 μg/mL) and ethidium bromide (EtBr) used as a positive control

Discussion

There are two major problems that impede increasing the efficiency of treatment for cancer patients:(1) non-addressed actions of many anticancer drugs that lead to general toxicity and severe negative side effects due to damage of normal tissues and organs; (2) rapid (6–12 months) development of resistance of tumor cells to applied anticancer drugs that leads to a loss of efficacy of drug action. In addition, there is a third, technical problem of poor water solubility of many anticancer drugs. However, binding these drugs with specific carriers of the nanoscale size can help to solve the solubility problem.

In this study, we addressed the first and third of the above-mentioned problems by (a) selecting novel synthetic substances (4-thiazolidinone derivatives:Les-3288 and Les-3833) that possess both high anticancer activity and less general toxicity in tumor-bearing animals [7, 8, 23] and (b) applying a novel drug delivery platform that provides stable water-based forms of the complexes of the water-insoluble 4-thiazolidinone derivatives with a PEGylated polymer.

The 4-thiazolidinone derivatives—Les-3288 and Les-3833—are heterocyclic compounds with molecular mass of 400–600 Da. These and other related derivatives were synthesized at Lviv National Medical University (Ukraine), and most of them have been tested under the Developmental Therapeutic Program at the National Cancer Institute in Bethesda, MA, (USA) [35,36,37,38]. Based on these evaluations of their antineoplastic activity, the most promising substances were selected for further study of the mechanisms of their cytotoxic action and anticancer effects [25]. Les-3833 demonstrated high toxicity towards B16F10, WM793, and SK-Mel-28 melanoma cells, and against human lung A549, breast MCF-7, colon HCT116, and ovarian SKOV3 cancer cells and leukemia cells (L1210, Jurkat, HL-60 lines) [23, 25, 39]. Les-3288 and Les-3833 showed high toxicity against mammalian glioma cells (C6, U251, U373 lines) [23, 24].

Earlier, we found that Les-3288 and Les-3833 were the most toxic among the other studied 4-thiazolidinone derivatives towards rat glioma C6 cells and human glioblastoma U251 cells [23, 24], although their application for cell treatment was very complicated due to their absolute insolubility in water. Only using DMSO with additional heating was effective for preparing a soluble form of the derivatives (see the “Materials and Methods” section). Thus, it was reasonable to search for a way of improving the delivery of these derivatives to target cells, and we used a synthetic PNC to accomplish this task. The complexation of the studied derivatives of 4-thiazolidinone with the applied PNC also enhanced the effectiveness of their antineoplastic action. The mechanisms of the pro-apoptotic action of these compounds were demonstrated using western blot and FACS analyses [23, 24]. It should be noted that in healthy experimental animals, Les-3288 and Les-3833 produce much fewer negative side effects such as cardiotoxicity [40], hepatotoxicity [8], and nephrotoxicity [7] than the widely used anticancer chemotherapeutic agent Dox.

In a previous study, we showed that Les-3288 did not induce ROS production during its pro-apoptotic action in vitro and effects in experimental laboratory animals [23], while the action of Les-3833 led to such production; however, it was less than that of Dox. ROS are considered to be the main effectors of negative side effects of anticancer drugs, particularly Dox [41, 42]. Thus, the Les-3288 and Les-3833 compounds could be prospective anticancer drugs because they possess antitumor activity, but do not demonstrate general toxicity at the level characteristic for effective anticancer medicines such as Dox.

A great impediment to promoting Les-3288 and Les-3833 as anticancer drugs is their poor water solubility. This problem also exists for other anticancer medicines, such as taxols [2, 4]. For paclitaxel, this problem was solved by using a special oil for preparing the medicinal formulation [2, 4, 43]. However, specific delivery platforms are considered to be more promising for creating “smart” drugs that possess effective action in the organism [3, 13]. Specific functional polymer surfactants with block, comb-like, and block/branched architectures, including PEG-containing ones, as well as derived water forms have been developed and proposed for application as carriers for the delivery of Dox [26, 27] and the metal chemotherapeutic agent KP-1019 [10] at the Department of Organic Chemistry of Lviv Polytechnic National University.

Since the 4-thiazolidinone derivatives are water-insoluble compounds, DMSO was used to prepare their water-soluble forms. To avoid the negative consequences of using a toxic solvent like DMSO, we complexed these derivatives with the above-noted PNC. Highly dispersed and stable nanoscale water dispersions of these substances were obtained and applied for treatment of rat glioma C6 cells.

The use of complexes of 4-thiazolidinone derivatives with the PNC led to reduced general toxicity of these compounds in experimental animals. Earlier, we found that when experimental antitumor agents (Les-3288, Les-3833, Les-3882) were applied in a complex with a synthetic polymeric carrier, their action was accompanied by much smaller changes in the biochemical parameters in blood serum that are characteristic for cardiotoxicity [40], hepatotoxicity [8], and nephrotoxicity [7], compared to those for Dox. Thus, complexation of these antitumor agents with the PNC and their application in the form of water-soluble stable delivery systems reduces their toxic effects in experimental animals, compared with the action of these substances in a free form [44]. Several systems for paclitaxel delivery have been proposed including polymeric nanoparticles, lipid-based formulations, polymer conjugates, inorganic nanoparticles, carbon nanotubes, nanocrystals, and cyclodextrin nanoparticles [43].

Thus, complexation of Les-3288 with the PNC significantly increased the pro-apoptotic activity of this experimental drug, thereby allowing us to advance it to pre-clinical studies on animal tumor models. Complexation of Les-3833 with the same type of nanocarrier was less efficient, and thus another type of polymer should be used.

The developed water-based forms of the complexes, as well as the free derivatives, were applied for treatment of rat glioma C6 cells. It was found that these water-based forms of the complexes of the PNC with the 4-thiazolidinone derivatives—Les-3288 and Les-3833—enhanced the pro-apoptotic action of these compounds. In another study, we demonstrated that the induction of apoptosis by Les-3288 was not accompanied by ROS induction, opposite to the action of Dox and Les-3833 [23]. Thus, high antitumor activity of Les-3288 together with its low general toxicity when targeting the malignancy in NK/Ly lymphoma-bearing mice suggest great potential for this experimental anticancer compound. The presented data also predict the potential usefulness of Les-3288 and Les-3833 compounds complexed with the PNC for glioma treatment.

Cell division, differentiation, and death are principal physiological processes that regulate tissue homeostasis in multicellular organisms. A disruption of genomic integrity and impaired regulation of cell death may both lead to uncontrolled cell growth. A compromised cell death process can also promote genomic instability. It is becoming clear that dysregulation of the cell cycle and cell death processes plays a pivotal role in the development of major disorders such as cancer, cardiovascular disease, infection, inflammation, and neurodegenerative diseases [45].

We have found that some of the 4-thiazolidinone derivatives, namely, Les-3288 and Les-3833, were even more effective than Dox in the induction of apoptosis in rat C6 glioma and human U251 glioblastoma cells in vitro [23, 24]. It should be noted that brain tumors belong to malignancies that are the most resistant to applied chemotherapies, and survival rates in patients with these tumors are extremely low [44].

It has been shown that the complexation with this PNC enhanced the pro-apoptotic action of 4-thiazolidinone derivatives (Les-3288 and Les-3833) [44]. In another study [23], we demonstrated that the induction of apoptosis by Les-3288 was not accompanied by ROS induction, opposite to the action of Dox and partially opposite to that of Les-3833. These data could explain the lower general toxicity of Les-3288 compared with that of Dox [23, 24].

The results of FACS analysis are in agreement with the suggestion that an enhancement of the pro-apoptotic action of the studied 4-thiazolidinone derivatives is due to their complexation with a novel PNC. Such complexation of Les-3288 and Les-3833 increased the ratio of pre-G1 cells in the population of treated rat glioma C6 cells, decreased the ratio of G1 cells, and increased the action of G2/M cells. These data suggest an enhancement of both cytotoxic action (apoptosis) and cytostatic action (block in G2/M phase of cell cycle) of the studied derivatives complexed with the PNC. In general, the apoptosis mechanism of glioma cell killing by the Les-3288 + PNC complex and the Les-3833 + PNC complex was demonstrated by the results of FACS analysis of the appearance of Annexin V single-positive rat glioma C6 cells.

Involvement of the apoptosis mechanisms in the action of the studied 4-thiazolidinone derivatives was also confirmed by the results of the DNA comet assay. It was found that the complexation of Les-3833 with the PNC led to a decrease (compared to free Les-3833) in the ratio of rat glioma C6 cells in “0” DNA comets and sіmultaneous increase in the ratio of “1,” “2,” and “3” comets (3-h treatment of cells). At 6 h of treatment by the Les-3833+PNC complex, there was a large increase (compared to free Les-3833) in the ratio of “1” comets, an increase in the ratio of cells with the most expressed DNA damage (“4” comets), and a decrease in the ratios of “2” and “3” comets. It should be noted that the DNA damaging effects of Les-3288 and Les-3833 measured at 6 h was higher than such effects of complexes of these derivatives with the PNC (see Table 3). A possible explanation here could be a very high level of the DI at 6 h action of studied agents that is even higher than the level of that indicator found for the action of Dox (Table 3). There was no time dependence in the action of the PNC, while a significant time-dependent (from 3 to 6 h) increase (from 41.2 + 0.40 to 185.2 + 0.23) in the DI was detected for the action of Dox.

Thus, a complexation of 4-thiazolidinone derivatives—Les-3288 and Les-3833—with a PNC enhanced the cytotoxic effect of these compounds towards rat glioma C6 cells, and that effect was achieved through the apoptosis mechanisms including DNA damage observed as DNA comets in the electrophoresed cells.

We used the DNA intercalation test to investigate the direct targeting of DNA by the 4-thiazolidinone derivatives and revealed only weak replacement of methyl green dye (15% and 10% replacement for Les-3288 and Les-3833, respectively) that intercalated into the DNA of salmon sperm, compared to the replacement by EtBr, used as a positive control (70%).

Thus, conducting the cytotoxicity experiments in vitro with the 4-thiazolidinone derivatives—Les-3288 and Les-3833—was more convenient due to using the water-based forms of the 4-thiazolidinone derivatives—Les-3288 and Les-3833—complexed with the novel PNC. Besides, we have demonstrated that the novel synthetic 4-thiazolidinone derivatives (Les-3833 and Les-3288) possessed treatment effects i n vivo towards NK/Ly lymphoma grafted to BALB/C mice, and those effects were comparable to such effects of Dox. At the same time, the action of these derivatives led to much fewer negative side effects measured as changes in the number of erythrocytes, neutrophils, and lymphocytes in the animals’ blood and the activity of aspartate and alanine aminotransferases in their blood serum, compared with the action of Dox.

結論

The complexation of Les-3288 and Les-3833 with the PEG-containing PNC enhanced their cytotoxic action towards rat glioma C6 cells. The cytotoxic action was realized by apoptotic mechanisms and confirmed by FACS analysis as the presence of the pre-G1 fraction in the rat glioma C6 cells and of Annexin V-single-positive cells. DNA comet analysis revealed single-strand brakes in the nuclear DNA of treated glioma C6 cells that probably was not caused by the intercalation of the studied compounds into the DNA molecule. The Les-3288 and Les-3833 stabilized by the amphiphilic PEG-containing PNC resulted in the water-based forms and provided enhancement of their antitumor effect in vitro in comparison with the free form of the drugs.

略語

DMSO:

ジメチルスルホキシド

Dox：

ドキソルビシン

EtBr:

Ethidium bromide

FACS:

Fluorescence-activated cell sorting

ROS:

活性酸素種

PBS：

リン酸緩衝生理食塩水

PEG：

ポリエチレングリコール

PI：

ヨウ化プロピジウム

PNC:

Polymeric nanocarrier

SEM：

走査型電子顕微鏡

TEM：

透過型電子顕微鏡

VEP:

2-Tret-butylperoxy-2-methyl-5-hexen-3-in