Novel platinum nanoclusters (Pt NCs) induce mitochondrial apoptosis and damaging autophagy for the treatment of osteosarcoma—from the perspective of P53 mutation status in different cell lines

Introduction

Osteosarcoma (OS) is one of the most predominant primary malignant bone tumors in children and adolescents^1,2. Despite the implementation of in situ amputation and systemic adjuvant chemotherapy for certain patients, the 5-year survival rate can plummet to as low as 20% due to inadequate tissue response to chemotherapy or the emergence of chemotherapy resistance^3,4. Cisplatin, a widely utilized platinum-based anticancer medication, serves as a fundamental component of osteosarcoma chemotherapy. However, cisplatin may be prone to drug resistance due to its single-action pathway⁵. Studies have shown that cisplatin alone has a therapeutic effect on only 30% of osteosarcoma patients⁶. Hence, improving and developing efficacious platinum-based drugs through nanomedicine and other methods and elucidating their anti-cancer mechanisms are urgent challenges in the field of osteosarcoma research.

P53, being a prominent tumor suppressor gene, exhibits mutations, loss, or reduced expression in numerous malignant tumors, thereby influencing the initiation and progression of cancer⁷. Furthermore, distinct cell lines within certain cancers display significant variations in P53 expression, consequently resulting in substantial disparities in the efficacy of P53-dependent chemotherapy drugs and patient prognosis^8,9. For instance, the chemotherapeutic agent curcumin has been found to induce apoptosis in P53 wild-type (P53⁺) breast cancer cell lines MCF-7 and TR-7, thereby exerting an anti-cancer effect. However, curcumin does not exhibit any impact on P53-deficient (P53⁻) breast cancer cells, namely MDAH041 and TR-9, indicating the limited broad-spectrum anticancer efficacy of this drug¹⁰. This pattern is also observed in osteosarcoma, where the mutation frequency of P53 is notably high¹¹. Specifically, the extensively investigated cell line MG-63 exhibits a loss-of-function mutation in P53 (P53⁻), while the U2-OS cell line retains the wild-type P53 status (P53⁺)¹². Moreover, MG-63 and U2-OS exhibit disparities in terms of cell morphology, proliferation, invasion, migration, and intricate signaling pathways¹³. Consequently, it is plausible that the administration of a particular anticancer drug, particularly one reliant on P53, may yield dissimilar outcomes when applied to these distinct cell types. Consequently, numerous investigations pertaining to the advancement of novel anti-osteosarcoma medications frequently undertake a collective examination of two or more cellular varieties encompassing MG-63 and U2-OS^11,14. These studies aim to elucidate the resemblances and disparities in the drug-induced impacts on the malignant conduct of these two cell types, thereby facilitating the exploration of the drug’s wide-ranging anti-cancer efficacy and potentially unraveling the intricate interplay between the drug, osteosarcoma, and P53.

Nanomedicine has been increasingly recognized due to its advantages in drug delivery, tumor eradication, and reversal of drug resistance brought about by its inherent nano-characteristics, different ligands, or processing techniques^15,16. Our research team endeavored to create a novel form of platinum-based chemotherapy medication utilizing nanotechnology. Specifically, we developed novel platinum nanoclusters (Pt NCs) and successfully demonstrated their remarkable efficacy in combating small cell lung cancer, leukemia, ovarian cancer, and even cisplatin-resistant ovarian cancer^17,18,19,20. Pt NCs have demonstrated the potential to inhibit the malignant characteristics of cisplatin-resistant ovarian cancer by potentially activating the PI3K/AKT/mTOR signaling pathway, inducing apoptosis, and promoting autophagy¹⁹. Considering that Pt NCs, a new platinum-based chemotherapy drug improved based on nanomedicine, has many advantages over cisplatin, a traditional osteosarcoma treatment drug, such as multiple action states and pathways and low toxic side effects, this study decided to explore the therapeutic potential of Pt NCs in osteosarcoma. Additionally, the relationship between osteosarcoma, P53 mutation, dysregulated apoptosis, and autophagy is intricate. Hence, it is imperative to investigate the potential of Pt NCs to operate in osteosarcoma cases characterized by varying P53 states, as well as to elucidate the intricate mechanisms underlying their association with apoptosis and autophagy.

This study aims to investigate and elucidate three key issues pertaining to osteosarcoma chemotherapy, utilizing two osteosarcoma cell lines, MG-63 (P53⁻) and U2-OS (P53⁺), with distinct P53 gene states. Firstly, the study seeks to determine the anticancer effect of Pt NCs on osteosarcoma. Secondly, it aims to ascertain whether there are any disparities in the anticancer effect of Pt NCs on osteosarcoma between the two aforementioned cell types. Lastly, the study endeavors to investigate the intricate underlying mechanism of Pt NCs in osteosarcoma, with the objective of identifying a dependable candidate for osteosarcoma chemotherapy.

Materials and methods

Cell culture

The primary focus of this study involved the utilization of two human osteosarcoma cell lines, namely MG-63 (with a deletion in the P53 gene, P53⁻) and U2-OS (with a wild-type P53 gene, P53⁺). In certain experiments, human bone marrow mesenchymal stem cell lines (hBMSC) were employed as control cells. All cell lines were procured from the Cell Center of the Chinese Academy of Sciences. MG-63 were cultured in DEME medium supplemented with 10% FBS, while U2-OS cells were cultured in McCoy’s 5A medium supplemented with FBS. Furthermore, they were maintained in a 5% CO₂ incubator at a temperature of 37 °C and subjected to trypsin digestion at the appropriate time for passaging. The aforementioned appropriate cells were employed in the subsequent series of experimental investigations.

Confocal microscopy confocal imaging

Different concentrations of Pt NCs (0 μg/mL, 0.1 μg/mL, and 0.15 μg/mL) were introduced into the MG-63, U2-OS, and hBMSC cells for co-culturing once the cell density reached the appropriate level. Following that, the process of cell fixation, washing, antibody incubation, and confocal imaging (LSM 830, Zeiss, Oberkochen, Germany) was carried out. These procedures were performed based on previous studies conducted by our research group¹⁹. This approach allowed for the observation of the ability of Pt NCs to enter both osteosarcoma cells and normal control cells. Similar procedures were carried out, and the viability of the respective cells was ultimately assessed using conventional light microscopy.

CCK-8 assay

The CCK8 assay was employed to assess the impact of Cisplatin and Pt NCs on the viability of MG-63 and U2-OS cells. The cells were seeded in 96-well plates at appropriate densities, and varying concentrations of Cisplatin and Pt NCs (ranging from 0 to 0.8 μg/mL) were subsequently introduced after cell adhesion. Following a 24-h co-culture period, the cells were treated with CCK8 reagent and incubated. The absorbance at 450 nm was then measured using an enzyme marker (PerkinElmer, USA) to determine the absorbance and calculate cell viability.

EdU immunofluorescence

The proliferation capacity of MG-63 and U2-OS cells was assessed using the EdU immunofluorescence assay. A suitable quantity of cell suspension was introduced onto slides in 6-well plates. Following cell adhesion, specific concentrations of Pt NCs were introduced for co-culture. Subsequently, cell fixation, washing, blocking, antibody incubation, and staining followed standard procedures. The Image J software (v1.8.0) was employed to quantify the proportion of cells in a proliferative state.

Clonogenesis assay

The cells MG-63 and U2-OS were cultured in 6-well culture dishes at a concentration of 200 cells/well, respectively. Subsequent steps of cell co-culture, colony observation, staining, washing, and drying were performed following the methodology established in a previous study conducted by our research group¹⁹. Subsequently, the shape, density, and quantity of colonies were documented and captured through photography, serving as indicators of the cells’ clonogenic potential. The number of colonies formed was quantified and compared using appropriate software, which is also capable of assessing clonogenicity.

Transwell assay

Cells (MG-63 and U2-OS) were inoculated in 200 μl of serum-free medium at a concentration of 1 × 10⁵ cells. Subsequently, the subsequent steps of cell incubation, cell transfer observation, cell fixation, staining, photography, and cell observation were conducted following the methodology established in our previous study¹⁹. Ultimately, the number of cells migrating from the upper Transwell chamber to the lower Transwell chamber was captured and documented, serving as an indicator of the cells’ migratory capability.

Wound healing assay

The 6-well culture dish was inoculated with the appropriate quantity of exponentially growing cells. Cell culture was conducted until the confluence reached 80%. A linear wound was created in the center of the culture dish using a pipette tip (200 μl). Non-adherent cells were removed by washing with PBS buffer. Subsequently, cells were cultured using a serum-free medium supplemented with specific concentrations of Pt NCs. The width and closure of the wound at the identical site were observed and documented at 0 and 24 h, respectively. The percentage of wound healing width serves as an indicator of the migratory capability of the cells.

Flow cytometry for cell cycle and apoptosis analysis

Cell cycle and apoptosis were assessed using flow cytometry, specifically through annexin V-PI staining. The subsequent steps of cell co-culture, washing, Annexin V/PI staining, flow cytometry, and cell observation were performed following the methodologies established in previous studies conducted by our research group¹⁹. Distinct life states were represented by cells in different regions, with Q1 indicating cell death, and Q2 + Q4 representing cell apoptosis. Conversely, Q3 denoted cell survival. The respective percentages of cells in each region were recorded and subjected to analysis. Additionally, the percentages of cells in different phases (G1, S, and G2) of the cell cycle were also recorded.

Western bolt

The standard procedure of the Protein Extraction Kit (Yarse) was followed to extract total protein. Protein concentrations were determined using the BCA method, and the denatured proteins were subsequently stored at −80 °C. The expression levels of the P53 protein, apoptosis-related proteins (BAX, Caspase 3, and Bcl-2), DNA damage-related protein (γH2A.X), and autophagy-related proteins (LC3B) were assessed in different groups. Gels were prepared utilizing a one-step kit (Yarse), followed by the loading of samples and electrophoresis. Subsequently, the bands were transferred onto PVDF membranes (Bio-Rad) and these membranes were subsequently blocked with BSA. The primary antibody, bound to the membrane, was then incubated overnight and subsequently washed. Following this, the membrane was incubated with the secondary antibody. The ultimate detection was carried out using the ECL (Y-aKinase) chemiluminescence kit. GAPDH was employed as an internal reference. The corresponding grayscale values were measured using Image J software (v1.8.0).

Xenograft tumor experiment in nude mice

Following a 7-day period of acclimation to the environment, 5-week-old immunodeficient nude mice were subjected to the establishment of a tumor-bearing model by injecting 200 μL of osteosarcoma cell suspension (143B, 2 × 10⁷ cells/mL) onto their right abdomen and back. All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act. The Ethics Committee of the Third Affiliated Hospital of Zhengzhou University granted approval for the relevant animal experiments. The growth of the tumors in the nude mice was monitored, and their body weight was regularly measured. Additionally, a vernier caliper was employed to record the long diameter (a) and short diameter (b) of the tumors, enabling the subsequent calculation of tumor volume (V = a × b²/2). When the tumor size approached approximately 20 mm³, the nude mice were partitioned into three groups, each consisting of five mice. Intraperitoneal injections of different substances were administered: the PBS group received 100 μL of PBS, the cisplatin group received 100 μL of cisplatin at a concentration of 4 mg/mL, and the Pt NC group received 100 μL of Pt NC at a concentration of 1 μg/mL. A total of three injections were administered over a span of seven days. After three weeks, the nude mice were euthanized through neck dislocation, and the tumors, heart, liver, spleen, lung, kidney, and other visceral tissues were extracted and photographed. The alterations in tumor volume and body weight of the nude mice were documented and computed. The visceral tissues, including the heart, liver, spleen, lung, and kidney, were subjected to embedding, sectioning, and staining with HE in order to assess the histotoxicity of the drug.

Detection of mitochondrial membrane potential

The detection of early apoptosis was conducted by utilizing the measurement of mitochondrial membrane potential (MMP). To detect MMP, the fluorescent dye JC-1 was employed. The cell suspension was transferred to a six-well plate at a suitable concentration, followed by a thorough washing step. Subsequently, the JC-1 staining working solution was added, and the plate was placed in a cell culture incubator set at 37 °C for a duration of 20 min. Finally, the cells were washed with JC-1 staining buffer. Lastly, the specimen should be examined using a fluorescence microscope. The presence of red-fluorescent “Aggregated” indicates elevated levels of MMP, while the presence of green-fluorescent “Monomers” signifies lower MMP. The progressive increase in the proportion of green fluorescence signifies the decline in MMP, which serves as a significant occurrence during the initial phase of mitochondrial apoptosis.

Autophagic flux detection by mRFP-GFP-LC3 adenovirus

The detection of autophagy and autophagic flux can be achieved through the utilization of the mRFP-GFP-LC3 adenovirus. Specifically, the mRFP component serves the purpose of tracking and labeling LC3, whereas the green fluorescence emitted by GFP is influenced by autophagy lysosomes, resulting in the manifestation of red fluorescence. The cell suspension should be transferred to a six-well plate at the appropriate concentration, followed by the addition of mRFP-GFP-LC3 adenovirus for transfection. After 48 h, the cells should be fixed, and the slides should be mounted for observation under a fluorescent microscope. The observed red fluorescence corresponds to autolysosomes, yellow fluorescence (Merge) corresponds to autophagosomes, and an increase in both red and yellow fluorescence in each cell indicates enhanced autophagic flux.

Statistical analysis

Statistical analysis was primarily conducted using GraphPad Prism software (v9.0). The cell proliferation, migration, apoptosis, and protein expression of varying concentrations of Pt NCs (0 μg/mL, 0.1 μg/mL, and 0.15 μg/mL) were compared. Furthermore, a comparative analysis was conducted to assess the influence of P53 on the disparity in the aforementioned capabilities between MG-63 and U2-OS at specific concentrations of Pt NCs. The majority of the empirical findings were expressed as the mean value accompanied by the standard deviation. To ascertain any significant distinctions between the mean values of two sets of data, a Student’s t-test was employed. Similarly, one-way ANOVA was utilized to evaluate any statistical disparities between the mean values of three sets of data.

Results

This study aimed to investigate the therapeutic effect and potential regulatory mechanisms of Pt NCs on osteosarcoma both in vitro and in vivo. Additionally, the study examined the impact of Pt NCs on osteosarcoma cell lines with varying P53 expression status, namely MG-63 (P53⁻) and U2-OS (P53⁺).

Pt NCs demonstrated a significant inhibitory effect on the cell viability, cell proliferation, and colony formation of osteosarcoma

The preparation and characterization of Pt NCs previously synthesized by our group were presented in the Supplementary Information (SI-1 and SI-2). The Pt NCs synthesized by our team earlier have their own fluorescence (specific green fluorescence). Consequently, as depicted in Supplementary Information (SI-3, Fig. S1A), confocal microscopy was employed to observe the entry of Pt NCs into osteosarcoma cells (U2-OS and MG-63) as well as normal control cells (hBMSC). The findings indicated that Pt NCs (specific green fluorescence) were localized in the vicinity of the nucleus (DAPI, specific blue fluorescence) or penetrated the nucleus in both groups of osteosarcoma cells. Conversely, Pt NCs exhibited negligible penetration into hBMSC. Subsequently, as depicted in Supplementary Information (SI-3, Fig. S1B), conventional light microscopy revealed that following Pt NCs treatment, the osteosarcoma cells in both groups exhibited a propensity towards spherical or fragmented morphology, indicating a decline in cellular activity. Moreover, this trend was observed to be contingent upon the concentration of Pt NCs. Conversely, such an effect was not prominently observed in hBMSC. Furthermore, the CCK8 results presented in Supplementary Information (SI-3, Fig. S1C) demonstrated a noteworthy suppressive impact of Pt NCs on the viability of osteosarcoma cells in both groups (p < 0.05), while exhibiting no significant influence on hBMSC (p > 0.05). The aforementioned findings indicated that Pt NCs possess the capacity to infiltrate osteosarcoma cells and exhibit fluorescent characteristics. Furthermore, Pt NCs exhibited potential cytotoxicity towards osteosarcoma cells while demonstrating lower toxicity towards normal cells as indicated by the cell morphology and CCK8 results.

Subsequently, the CCK8 assay results depicted in Fig. 1A demonstrated a substantial decline in cellular activity in both groups of osteosarcoma cells with increasing Pt NCs concentration, whereas the conventional chemotherapeutic drug cisplatin proved to be less effective (p < 0.05). This observation underscores the superiority of Pt NCs over cisplatin. Furthermore, the immunofluorescence results depicted in Fig. 1B, C demonstrate a decline in the proportion of cells in a proliferative state (EdU positive) as the concentrations of Pt NCs increase (MG-63, 44.09% > 35.05% > 31.67%, p < 0.05; U2-OS, 60.73% > 55.16% > 49.92%, p < 0.05). Additionally, the findings from the clone formation analysis in Fig. 1D indicate a reduction in both the size and quantity of formed clones with escalating concentrations of Pt NCs (MG-63, 78.00 > 47.00 > 3.00, p < 0.05; U2-OS, 115.30 > 80.00 > 36.33, p < 0.05).

Novel platinum nanoclusters (Pt NCs) induce mitochondrial apoptosis and damaging autophagy for the treatment of osteosarcoma—from the perspective of P53 mutation status in different cell lines — **Fig. 1: Inhibition of proliferation and colony-forming ability of osteosarcoma cells by Pt NCs.**

Pt NCs exerted inhibitory effects on cell migration, while also promoting apoptosis in osteosarcoma

Subsequent to this, the Transwell findings depicted in Fig. 2A indicated a noteworthy reduction in the number of cells that ultimately migrated to the lower compartment as the concentration of Pt NCs escalated (MG-63, 30.00 > 10.67 > 2.00, p < 0.05; U2-OS, 59.33 > 33.33 > 16.67, p < 0.05). Furthermore, the outcomes of the scratch healing assay presented in Fig. 2B demonstrated a deceleration in the process of scratch healing as the concentration of Pt NCs heightened (MG-63, 94.50 > 50.93 > 35.08, p < 0.05; U2-OS, 96.90 > 61.92 > 48.41, p < 0.05). Collectively, these findings strongly imply that Pt NCs possess the capability to impede the migration of osteosarcoma cells. Additionally, the flow cytometry findings in Fig. 3A indicated an increase in the percentage of cells in the apoptotic state (Q2 + Q4) with escalating Pt NCs concentrations (MG-63, 2.68 < 26.37 < 66.32, p < 0.05; U2-OS, 2.94 < 25.72 < 50.89, p < 0.05).

**Fig. 2: Inhibition of the migration ability of osteosarcoma cells by Pt NCs.**

**Fig. 3: Promotion of apoptosis in osteosarcoma cells by Pt NCs.**

The Western blotting results depicted in Fig. 3B, C indicated that as the concentrations of Pt NCs increased, there was an absence of P53 protein expression in MG-63 cells. Conversely, the expression of P53 protein in U2-OS cells exhibited a gradual increase (0.57 < 0.69 < 0.96, p < 0.05). These findings provided evidence for the deletion of P53 expression (P53⁻) in MG-63 cells and suggested that Pt NCs have the ability to enhance P53 protein levels in U2-OS cells (P53⁺). Following this, the concentrations of Pt NCs exhibited a direct correlation with the upregulation of apoptosis-promoting proteins [BAX (MG-63, 0.71 < 0.98 < 1.19, p < 0.05; U2-OS, 0.63 < 1.02 < 1.16, p < 0.05); Caspase-3 (MG-63, 0.81 < 0.94 < 1.13, p < 0.05; U2-OS, 0.61 < 0.81 < 1.00, p < 0.05)] and concurrent downregulation of apoptosis-inhibiting proteins [Bcl-2, (MG-63, 0.95 > 0.76 > 0.67, p < 0.05; U2-OS, 1.10 > 0.83 > 0.66, p < 0.05)].

The in vivo suppression of osteosarcoma by Pt NCs

Following an examination of the anticancer properties of Pt NCs on osteosarcoma cells, we proceeded to investigate their effects through xenograft experiments conducted on nude mice. The findings presented in Fig. 4B indicated that the administration of Pt NCs did not result in significant harm to the heart, liver, spleen, lung, and kidney tissues of the nude mice. Moreover, when considering the outcomes pertaining to the normal control cells (hBMSC) depicted in Supplementary Information (SI-3, Fig. S1), it could be inferred that Pt NCs do not exhibit evident toxicity towards normal cells or tissues, thus signifying a substantial advantage. Furthermore, of equal significance, the outcomes depicted in Fig. 4C demonstrated substantial inhibition of transplanted tumor growth in nude mice by Pt NCs (p < 0.05), surpassing the efficacy of the conventional drug cisplatin. Consequently, the in vivo experiments conducted on nude mice provided robust confirmation of the selective cytotoxicity and tissue toxicity of Pt NCs toward osteosarcoma at the animal level.

**Fig. 4: Antitumor effect and tissue toxicity of Pt NCs against osteosarcoma in vivo.**

Differential anti-tumor effects of Pt NCs on MG-63 (P53⁻) and U2-OS (P53⁺)

In the aforementioned findings depicted in Figs. 1–3, it was observed that identical concentrations of Pt NCs exhibited varying anti-tumor effects on MG-63 and U2-OS cell lines, suggesting a potentially higher sensitivity of MG-63 cells to Pt NCs. Consequently, our endeavor was directed towards elucidating and substantiating this potential disparity. After further selecting the appropriate concentration of Pt NCs, a subsequent set of phenotypic experiments was conducted on the aforementioned cells. The results in Supplementary Information (SI-4, Fig. S2A) showed that the inhibitory effect of Pt NCs at the same concentration on the cell viability of MG-63 cells was significantly higher than that of U2-OS cells (0.1 μg/mL, 23.92% > 6.15%, p < 0.05; 0.15 μg/mL, 70.67% > 16.92%, p < 0.05). The results in Supplementary Information (SI-4, Fig. S2B) showed that the inhibitory effect of Pt NCs at the same concentration on the percentage of cells in the proliferation phase of MG-63 cells was significantly higher than that of U2-OS cells (0.1 μg/mL, 20.21% > 9.19%, p < 0.05; 0.15 μg/mL, 28.00% > 17.82%, p < 0.05). The results in Supplementary Information (SI-4, Fig. S2C) showed that the inhibitory effect of Pt NCs at the same concentration on the clone-forming ability of MG-63 cells was significantly higher than that on U2-OS cells (0.1 μg/mL, 39.74% > 30.67%, p < 0.05; 0.15 μg/mL, 96.17% > 68.46%, p < 0.05). The results in Supplementary Information (SI-4, Fig. S2D) showed that the inhibitory effect of Pt NCs at the same concentration on the migration ability of MG-63 cells was significantly higher than that on U2-OS cells (0.1 μg/mL, 64.00% > 43.77%, p < 0.05; 0.15 μg/mL, 93.19% > 71.51%, p < 0.05). The results in Supplementary Information (SI-4, Fig. S2E) showed that the same concentration of Pt NCs had a significantly higher inhibitory effect on the wound healing rate of MG-63 cells than that of U2-OS cells (0.1 μg/mL, 46.10% > 36.09%, p < 0.05; 0.15 μg/mL, 62.89% > 50.03%, p < 0.05). The results in Supplementary Information (SI-4, Fig. S2F) showed that the same concentration of Pt NCs promoted the apoptosis of MG-63 cells more significantly than that of U2-OS cells (0.15 μg/mL, 63.64% > 47.95%, p < 0.05). The comprehensive findings depicted in Supplementary Information (SI-4, Fig. S2) demonstrated that Pt NCs exhibited a more pronounced inhibitory effect on the proliferation and migration of MG-63 (P53⁻) cells compared to U2-OS (P53⁺) cells. Furthermore, Pt NCs also exhibited a more substantial promotion of apoptosis in MG-63 cells.

The potential mechanism underlying the antitumor effect of Pt NCs on MG-63 cells

In order to explore the detailed mechanism of the anti-tumor effect of Pt NCs on MG-63, the cell cycle, which is often studied, was first used as the entry point for research. The flow cytometry results depicted in Fig. 5A demonstrated that Pt NCs have the capability to induce cell cycle arrest in MG-63 cells, specifically in the G1 phase (40.22% < 47.66%, p < 0.05). The cell cycle arrest is closely associated with DNA damage. Furthermore, the western blot results presented in Fig. 5B revealed a substantial upregulation in the expression of γH2A.X (1.00 < 1.23, p < 0.05), a protein characteristic of DNA damage, upon treatment with Pt NCs. These findings strongly implied that Pt NCs possess potential anticancer properties through their ability to promote DNA damage and arrest the cell cycle.

**Fig. 5: Effects of Pt NCs on cell cycle, DNA damage, and mitochondrial membrane potential in MG-63 (P53⁻).**

Moreover, the findings from the analysis of mitochondrial membrane potential in Fig. 5C demonstrated a progressive increase in the proportion of green fluorescent “Monomers” (61.66% < 84.55%, p < 0.05) in response to Pt NCs, suggesting their ability to diminish the mitochondrial membrane potential. This decline in mitochondrial membrane potential serves as a significant indicator of early apoptosis. This finding, in conjunction with the outcomes of apoptosis flow cytometry and Western blot analysis of apoptosis characteristic proteins depicted in Fig. 3, implied that Pt NCs exert a modulatory influence on the BAX/Bcl-2 protein ratio, diminish the mitochondrial membrane potential, thereby triggering the caspase cascade reaction and activating Caspase-3 protein, ultimately leading to apoptosis via the mitochondrial pathway.

In addition, autophagy is a topic that warrants further investigation in the context of anti-cancer strategies. Initially, the findings depicted in Fig. 6A, which were derived from the assessment of mRFP-GFP-LC3 adenovirus’s autophagic flow, indicated a substantial rise in the number of red and yellow fluorescence, denoting an augmented presence of autolysosomes and autophagosomes (autolysosomes, 3.00 < 10.60, p < 0.05; autophagosomes, 2.93 < 9.83, p < 0.05), which suggested the enhancement of autophagic flow. Furthermore, the Western blot analysis depicted in Fig. 6B demonstrated that Pt NCs have the ability to enhance the expression levels of autophagy-associated proteins (LC3B). These observations implied that Pt NCs possess the potential to stimulate autophagy. Furthermore, autophagy can be categorized into two distinct types: damaging autophagy and protective autophagy. Both forms of autophagy serve unique functions and merit in-depth exploration. It is worth noting that 3-MA is a widely employed inhibitor of autophagy and its application is helpful to study autophagy. The Western blot analysis depicted in Fig. 6B demonstrated the inhibitory effect of 3-MA on autophagy in osteosarcoma (Fig. 6B, 3-MA vs NC, 0.77 < 1.00, p < 0.05), as well as its ability to impede autophagy induced by Pt NCs (Fig. 6B, 3-MA+ Pt NCs vs Pt NCs, 0.95 < 1.34, p < 0.05). Moreover, given the findings of this investigation indicating that Pt NCs have the potential to augment autophagy (Fig. 6B, Pt NCs vs NC, 1.34 > 1.00, p < 0.05) while concurrently impeding cell viability, it could be inferred that the autophagy triggered by Pt NCs may be damaging autophagy. Furthermore, the outcomes of apoptosis flow cytometry presented in Fig. 6C implied a potential link between the damaging autophagy induced by Pt NCs and mitochondrial apoptosis (Fig. 6C, Pt NCs vs NCs, 44.41 > 3.63, p < 0.05). In Fig. 6B, it was observed that there was no notable disparity in autophagy between the NC group and the 3-MA+Pt NCs group (Fig. 6B, 3-MA+ Pt NCs vs NCs, 0.95 < 1.00, p > 0.05). However, in Fig. 6C, it was evident that the level of apoptosis in the 3-MA+Pt NCs group was significantly greater than that in the NC group (Fig. 6C, 3-MA+ Pt NCs vs NCs, 32.98 > 3.63, p < 0.05). This finding suggested the potential of Pt NCs to induce both damaging autophagy and mitochondrial apoptosis. Furthermore, it implied that the damaging autophagy triggered by Pt NCs may concurrently facilitate mitochondrial apoptosis.

**Fig. 6: Effects of Pt NCs on autophagy in MG-63 (P53⁻).**

Hence, based on the above research results, it is reasonable to speculate that in osteosarcoma cells MG-63 with P53 mutation deletion (P53⁻), Pt NCs have the potential to exert an anti-cancer effect through the facilitation of DNA damage for cell cycle arrest, the promotion of BAX/Bcl-2/Caspase-3/mitochondrial apoptosis, and the induction of damaging autophagy. Furthermore, the observed damaging autophagy induced by Pt NCs may further enhance mitochondrial apoptosis. In addition, it could be reasonably speculated that in osteosarcoma cells U2-OS with normal P53 expression (P53⁺), Pt NCs may play an anticancer role by promoting BAX/Bcl-2/Caspase-3/mitochondrial apoptosis and inducing the P53 pathway. The above reasonable speculations about the mechanism of action of Pt NCs in osteosarcoma cells with different P53 expression states were recorded in Supplementary Information (SI-5, Fig. S3).

Discussion

Chemotherapy for osteosarcoma, especially platinum-based chemotherapy drugs, is prone to poor sensitivity or even drug resistance, which needs to be improved urgently²¹. The application of nanoscale drug enhancements holds significant potential for enhancing the effectiveness of chemotherapy²². Hence, this research aims to investigate the therapeutic impact and intricate mechanism of the novel platinum-based nanomedicine Pt NCs on osteosarcoma. Supplementary Information (SI-5, Fig. S3) represented our conjecture regarding the potential mechanism of Pt NCs in osteosarcoma, derived from the findings of this study.

In the realm of anticancer drug development, exploring the ability to eradicate tumors and their toxicity to normal cells is undoubtedly the most basic. Supplementary Information (SI-3, Fig. S1) showed the potential of Pt NCs as a new therapeutic drug for osteosarcoma to enter and eradicate tumors and have low toxicity to normal cells. In addition, the fluorescent characteristics of Pt NCs shown in Supplementary Information (SI-3, Fig. S1) may confer an advantageous diagnostic edge to chemotherapy drugs²³. Resistance to conventional drug cisplatin is a prevalent occurrence in numerous tumor types²⁴. Figure 1A indicated the superiority of Pt NCs in comparison to cisplatin. Furthermore, recent studies have demonstrated encouraging therapeutic prospects for Pt NCs in the context of cisplatin-resistant ovarian cancer and non-small cell lung cancer cells^17,19. It is imperative and fundamental to investigate the impact of chemotherapy agents on the malignant characteristics of osteosarcoma cells. Subsequently, Figs. 1–3 indicated that Pt NCs have the ability to impede malignant characteristics such as proliferation, migration, and colony formation while fostering apoptosis of osteosarcoma cells. The subsequent experiment presented in Fig. 4 involved nude mouse xenograft tumor transplantation and yielded results that confirmed the in vivo inhibitory capability of Pt NCs against osteosarcoma. Besides, combined with the weak toxicity of Pt NCs to normal control cells hBMSC shown in Supplementary Information (SI-3, Fig. S1), it is suggested that the toxicity of Pt NCs to cells and tissues is not obvious, which is undoubtedly an advantage. Overall, the aforementioned outcomes substantiate the inhibitory impact of Pt NCs on osteosarcoma at both cellular and animal levels, thereby indicating its potential as a promising therapeutic agent for osteosarcoma.

Due to its status as one of the earliest identified tumor suppressor genes, P53 serves as a crucial factor for certain chemotherapeutic drugs. These drugs function by either reinstating the activity of P53 or enhancing its expression, offering a stable and feasible approach²⁵. However, it is important to acknowledge the potential drawbacks associated with these drugs. The expression of the P53 gene can exhibit significant variability in certain tumors, thereby resulting in a considerably low sensitivity of P53 mutant (P53⁻) cells toward P53-dependent chemotherapy drugs. Consequently, this limitation restricts the widespread applicability of P53-dependent chemotherapy drugs^10,26. The MG-63 osteosarcoma cell line is characterized by a loss of P53 mutation (P53⁻) and a higher degree of malignancy, whereas the U2-OS cell line is characterized by a wild-type P53 (P53⁺)¹¹. Therefore, investigating the impact of Pt NCs on osteosarcoma cells with different P53 genotypes will not only contribute to the determination of their broad-spectrum anticancer effects but also facilitate a more comprehensive understanding of the underlying mechanisms of Pt NCs. In this study, Fig. 3B, C confirmed the absence of P53 gene expression in MG-63 cells, while indicating that Pt NCs have the potential to enhance the expression of P53 in U2-OS cells. Subsequently, Supplementary Information (SI-4, Fig. S2) indicated that, compared with U2-OS, Pt NCs have stronger effects on MG-63 (P53⁻) in inhibiting proliferation, migration, and colony formation while promoting apoptosis. These outcomes implied that Pt NCs possess a wide-ranging anticancer activity against various osteosarcoma cell lines. Furthermore, the anticancer efficacy of Pt NCs remained unaffected by the absence of P53 in the more malignant MG-63 cells (P53⁻) and is particularly potent against MG-63 cells in comparison to U2-OS cells, thereby offering a potential avenue for further investigation into the underlying mechanisms.

The deletion mutations of P53 are prevalent in osteosarcoma cells, leading to significant variations in the cell signaling pathways among osteosarcoma cells with different P53 expression states. Notably, MG-63 cells harboring deletion mutations of P53 (P53⁻) exhibit a higher degree of malignancy²⁷. Consequently, we have selected MG-63 cells (P53⁻) as the subjects for further investigation into the underlying mechanisms.

DNA damage and cell cycle arrest play significant roles in the decrease of cell proliferation²⁸. Figure 5A, B indicates that Pt NCs have the potential to induce DNA damage and halt the cell cycle of osteosarcoma cells during the G1 phase. Consequently, it could be hypothesized that Pt NCs possess the ability to impede the proliferation of osteosarcoma cells through the facilitation of DNA damage and cell cycle arrest. On the other hand, apoptosis, which is classified as type I cell death, plays a significant role in the process of programmed cell death²⁹. The expression of key proteins involved in the mitochondrial pathway of apoptosis was depicted in Fig. 3B, C. Furthermore, Fig. 5C indicated that Pt NCs have the potential to induce a reduction in mitochondrial membrane potential (MMP), a crucial event in mitochondrial permeability alteration and subsequent cell death. Hence, it is plausible to hypothesize that Pt NCs may enhance the BAX/Bcl-2 ratio, resulting in a decrease in mitochondrial membrane potential and an increase in mitochondrial membrane permeability. Consequently, this would initiate the caspase cascade reaction and activate Caspase-3, ultimately leading to cellular apoptosis. In other words, Pt NCs possess the capability to promote BAX/Bcl-2/Caspase-3/mitochondrial apoptosis.

Autophagy, a form of programmed cell death known as type II cell death, exhibits an intricate and intimate association with the initiation and progression of cancer, warranting further investigation³⁰. Nanoparticles may be engulfed by cells as foreign objects due to their unique nanometer size, thereby triggering autophagy³¹. However, it is important to note that autophagy operates as a two-sided mechanism, capable of inducing cell demise (referred to as damaging autophagy) as well as facilitating cell survival (known as protective autophagy)³². Regarding Pt NCs, they potentially contribute to the promotion of protective autophagy in small-cell lung cancer but may play a role in damaging autophagy in ovarian cancer^19,33. Hence, an in-depth investigation was conducted to examine the correlation between Pt NCs and autophagy in osteosarcoma. Figure 6A, B indicates that Pt NCs possess the capability to induce autophagy in osteosarcoma. Given the observed anti-cancer properties of Pt NCs in this investigation, it is postulated that the autophagy triggered by Pt NCs may be damaging autophagy. Upon careful examination of Fig. 6B, C, in conjunction with the application of an autophagy inhibitor (3-MA), it is hypothesized that Pt NCs might have the capacity to induce both mitochondrial apoptosis and damaging autophagy concurrently, and the damaging autophagy induced by Pt NCs could potentially facilitate the promotion of mitochondrial apoptosis simultaneously.

Reviewing the full text, this study investigated for the first time the anticancer effect of novel nanomedicine Pt NCs in osteosarcoma of different P53 genotypes, MG-63 (P53⁻) and U2-OS (P53⁺), and verified it at the animal level, and tried to elucidate its potential mechanism in MG-63 (P53⁻) cell cycle, apoptosis and autophagy.

Conclusion

Overall, for osteosarcoma cells of different P53 genotypes, MG-63 (P53⁻) and U2-OS (P53⁺), the novel platinum nanoclusters (Pt NCs) developed in this study could significantly inhibit proliferation, migration, colony formation and promote apoptosis, suggesting that the anticancer effect of Pt NCs is P53-independent. The animal experiment also confirmed the anti-tumor effect of Pt NCs in vivo. Pt NCs may potentially exhibit anticancer properties in the more sensitive MG-63 (P53⁻) cells through the facilitation of DNA damage to halt the cell cycle, stimulation of BAX/Bcl-2/Caspase-3/mitochondrial apoptosis, and initiation of damaging autophagy. Furthermore, the induction of damaging autophagy by Pt NCs could further enhance mitochondrial apoptosis.